Tissue-specific and pathogens-specific toxic agents, ribozymes, dnazymes and antisense oligonucleotides, and methods of use thereof

ABSTRACT

The present invention relates to the discovery, identification and characterization of toxic agents which are lethal to pathogens and methods for targeting such toxic agents to a pathogen or pathogen infected cells in order to treat and/or eradicate the infection. In particular, the present invention relates to toxic agents which target bacteria at different stages of the bacterial life cycle, which are delivered alone or in combination to bacteria or bacteria-infected cells. The invention relates to toxic agents which are lethal to diseased cells and methods for targeting such toxic agent to a diseased cell in order to treat and/or eradicate the disease. The present invention relates to promoter elements which are pathogen-specific or tissue-specific and the use of such promoter elements to achieve pathogen-specific or tissue-specific expression of the toxic agent(s) and/or ribozyme(s) of the present invention. Specifically, the invention relates to the delivery of one or more toxic gene products, antisense RNAs, or ribozymes, or combination thereof. The invention provides a novel system by which multiple pathogenic targets may be simultaneously targeted to cause the death of a pathogen, or cell infected with a pathogen. Further, the invention has important implications in the eradication of drug-resistant bacterium and bacterial pathogens. The invention provides a novel system by which multiple targets may be simultaneously targeted to cause the death of a diseased cell. The invention has important implications in the eradication of drug-resistant pathogens (such as antibiotic resistant bacteria) and drug-resistant diseased cells (such as drug-resistant cancer cells).

1. INTRODUCTION

[0001] The present invention relates to the discovery, identification,and characterization of toxic agents which are lethal to pathogens andmethods for targeting and delivering such toxic agents to a pathogen orpathogen infected cell in order to treat and/or eradicate an infection.In particular, the present invention relates to toxic agents whichtarget bacteria at different stages of the bacterial life cycle, whichare delivered alone or in combination to bacteria or bacteria-infectedcells. In particular, the invention relates to a phage delivery vehicleapproach for the treatment of bacterial infections in humans andanimals. The invention also relates to toxic agents which are lethal todiseased cells and methods for targeting such toxic agents to a diseasedcell in order to treat and/or eradicate the disease. The presentinvention relates to promoter elements which are pathogen-specific. Theinvention also relates to promoter elements which are used to achievepathogen-specific or tissue specific expression of the toxic agent(s)and/or ribozyme(s) of the present invention. Specifically, the inventionrelates to the delivery of one or more toxic gene products, antisenseRNAs, or ribozymes, or combination thereof. The invention provides anovel system by which multiple pathogenic targets may be simultaneouslytargeted in order to kill a pathogen or pathogen-infected cell or renderit less fit. Further, the invention has important implications in theeradication of drug-resistant bacterium and bacterial pathogens. Theinvention provides a novel system by which multiple targets may besimultaneously targeted to cause the death of a diseased cell or renderit less fit. The invention has important implications in the eradicationof drug-resistant pathogens (such as antibiotic resistant bacteria) anddrug-resistant diseased cells (such as drug-resistant cancer cells). Thepresent invention also relates to DNAzymes, antisense oligonucleotidesand ribozymes useful in pharmaceutical compositions for the treatment ofviral infections including papillomavirus and hepatitis B.

2. BACKGROUND

[0002] 2.1. Antimicrobial Agents

[0003] Infectious diseases sicken or kill millions of people each year.Each year in the United States alone, hundreds of thousands of peopleare infected with resistant bacterial strains that are no longertreatable with drugs like penicillin and vancomycin (Hiramatsu et al,1997, Morbidity and Mortality Weekly Report 46:624-26). Infectionsassociated with antimicrobial resistance include those acquired inhospitals (nosocomial), such as neumonia particularly in the young,elderly and immunocompromised), typhoid fever, acterial meningitis, andtuberculosis. Around the world, nearly 1.5 billion people carry varioustypes of the tuberculosis bacteria and depending on the country, up to40 percent have proven to be resistant to antibiotics (see, Boyce et al,1997, Epidemiology and prevention of nosocomial infections. In TheStaphylococcus in Human Disease. Crassly and Archer E's, ChurchillLivingston Inc., New York, N.Y.). It is estimated that in some developedcountries, up to 60% of all nosocomial infections result from bacteriaresistant to antibiotics. For example, Pseudomonas aeruginosa, is of themost common gram-negative bacterium associated with nosocomialinfections and outbreaks in burn units. Infections by this organism areassociated with high mortality (60%), which is attributed to the highintrinsic resistance of members of this genus to many structurallyunrelated antibiotics. Gram-positive bacteria also have a significantimpact on infectious diseases. For example, Staphylococcus aureus, is aGram-positive organism which is responsible for about 260,000 hospitalacquired infections in the United States which subsequently causesbetween 60,000 and 80,000 deaths annually (see, Boyce et al, supra).

[0004] Although, numerous antimicrobial therapies have been designed totarget one or several infectious agents, many therapies show varyingdegrees of success in eradicating infection. Only a very limited numberof new antibiotics have come onto the market in the last decade, yet thenumber of deadly bacteria that are resistant to these drug therapies hassoared. For example, vancomycin is one of the last effectiveantimicrobial available for the treatment of methicillin-resistant S.aureus infection (MRSA). However, vancomycin resistant isolates S.aureus have now emerged (Hiramatsu et al, 1997, Morbidity and MortalityWeekly Report 46:624-26). Additionally, the failure of many of thesetherapies to target specific infectious agents has lead to overuse orinappropriate use of the therapies, which in turn has lead to thedevelopment of drug resistant microbes. The development of drugresistance in many infectious agents has reduced the efficacy andincreased the risk of using the traditional antimicrobial therapies.

[0005] Accordingly, there is need in the art for novel molecules andnovel combinations of molecules that can act as lethal agents inbacteria and which may be delivered to a pathogen, without causingtoxicity to the infected host. Further, there is a need in the art fornovel methods of targeting particular species of pathogens while leavingthe host's beneficial flora intact. The present invention provides suchnovel products, therapeutics, and methods for delivery which may be usedas toxic agents against pathogens such as bacteria.

[0006]2.2. Antisense

[0007] Antisense technology seeks to use RNA molecules which arecomplementary to (or antisense to) a cellular RNA, for the purpose ofinhibiting a cellular RNA from being translated into the encodedprotein. In this way, the expression of a specific protein is targetedfor down regulation. However, a large number of difficulties exist inthe art surrounding antisense technology. Commonly, delivery of anexogenous antisense molecule to the target cell is difficult orimpossible to achieve. Further, antisense molecules do not consistentlylead to a decrease in protein expression. For example, it has been shownthat the expression of antisense RNA in transgenic mice did notinvariably lead to a reduction in target RNA molecules, and whenreduction in target RNA molecules did occur, it was not predictablyparalleled by a reduction in protein. Even when protein levels werereduced sometimes no biological effect was detected (Whitton, J. Lindsay“Antisense Treatment of Viral Infection” Adv. in Virus Res. Vol. 44,1994). Thus, there is a need in the art for a delivery system in whichantisense molecules may be efficiently delivered to a target cell suchas a bacterial pathogen.

[0008] 2.3. Ribozymes

[0009] A ribozyme is a catalytic RNA molecule that cleaves RNA in asequence specific manner. A key technical concern in the use ofribozymes as antimicrobial agents is that the ribozyme must beintroduced into and expressed by the targeted microbe so that theribozyme(s) can cleave the targeted RNA(s) inside the microorganism. Asecond important concern is the tight coupling of transcription andtranslation in microorganisms which can prevent binding to and cleavageof the bacterial RNA targets. Additionally, bacterial RNAs often have ashorter half life than eukaryotic RNAs, thus lessening the time in whichto target a bacterial RNA. The invention described herein addressesthese concerns and proves novel therapeutic treatments of bacterialinfections using combinations of ribozymes and toxic agents.

3. SUMMARY OF THE INVENTION

[0010] The present invention provides toxic agents and methods forspecifically targeting toxic agents to bacteria or bacteria-infectedcells or other pathogens. Toxic agents of the present invention aredirected to one or more targets and thus can be used alone or incombination to eradicate bacteria. The invention relates to the deliveryof toxic gene products or the combination of ribozymes and toxic geneproducts for the eradication of a pathogen or diseased cell.Specifically, the invention provides the delivery of one or more toxicproteins, antisense RNA, multi-ribozymes, or nucleic acids encoding thesame, or a combination thereof, to a cell, tissue, or subject containingan infectious bacteria or pathogen in order to eradicate such bacteriaor pathogen.

[0011] The present invention further encompasses the use of a toxicagent and/or ribozymes of the present invention for the treatment ofdisease, viral infection, parasitic infection and microbial infection.The present invention further relates to a method of treating a subjecthaving a proliferative disease of a specific tissue by inhibiting cellproliferation in the tissue, comprising administering to the subject atoxic agent and/or ribozyme operably linked to a tissue-specificpromoter sequence, which promoter is specific for the diseased tissue,and whereby the ribozyme and/or toxic agent encoded by the nucleic acidis expressed, cell proliferation is inhibited, and the proliferativedisease is treated.

[0012] The present invention further relates to a method of treating asubject having a pathogenic infection or disease, by inhibitingreplication of the pathogen, comprising administering to the subject atoxic agent and/or ribozyme operably linked to a pathogen-specificpromoter, whereby the ribozyme and/or toxic agent encoded by the nucleicacid is expressed, the pathogen is inhibited from replicating or iskilled or rendered less fit, and the infection or disease is treated. Inspecific embodiments of the invention, the toxic agents of the inventionare useful to treat microbial infections associated with severe burns,cystic fibrosis, cancer, or other immunocompromising conditions. Thepresent invention encompasses the toxic agent(s) and/or ribozyme(s) ofthe present invention in pharmaceutical formulations.

[0013] The present invention further encompasses the use of the toxicagents and/or ribozymes of the present invention for research andscreening purposes. In one embodiment of the present invention, theribozymes and/or toxic agents may be used to screen for viral,microbial, prokaryotic, or eukaryotic gene products or molecules to betargeted in order to effectively inhibit the selected virus or microbialagent or selected cell.

[0014] In yet another embodiment, the present invention relates to anovel vector encoding the toxic agent(s) and/or ribozyme(s). The novelvectors of the present invention may be used to engineer a wide varietyof toxic agents and/or ribozymes including, but not limited to,tissue-specific, pathogen-specific, promoter-specific, antimicrobialspecific, antiviral specific, anticancer specific, antitumor specific,or target-specific.

[0015] In one embodiment, the invention relates to toxic agents whichspecifically target gene products essential for the survival or lifecycle of a pathogen (such as replication, packaging, etc). In oneembodiment, the present invention relates to naturally occurringbactericidal addiction system toxins which have been modified to beexpressed in the absence of their corresponding addiction systemantidote. In another embodiment, the present invention relates tonaturally occurring addiction system toxins which have been modified tobe expressed at higher levels than their corresponding addiction systemantidote. In one example, an addiction system toxin (e.g., doe, chpBK,kicB, or gef) is used as a toxic agent and is uncoupled from itsantidote. In specific embodiments, the invention provides for deliveryof toxic agents such as bactericidal proteins (or nucleic acids encodingsuch toxic agents) by a bacteriophage delivery system. In other specificembodiments, the invention provides novel transfer plasmids encodingtoxic agents which may be used in combination with a bacteriophagedelivery system in order to treat a bacterial infection in a host.

[0016] The invention also relates to antisense RNA which targetsessential nucleotide sequences, such as DicF1 or a DicF1-like antisensemolecule that specifically target a nucleotide sequence encoding aprotein essential for replication or survival. Further, the inventionrelates to modified antisense structures with increased stability whichact as lethal agents when expressed in bacteria. The invention alsorelates to toxic sense molecules designed to target essential antisensemolecules.

[0017] The present invention relates to multi-ribozymes and their use totarget RNA in a tissue-specific or pathogen-specific manner for thetreatment of disease (such as pathogen infection or cancer). Theinvention provides multi-ribozymes containing one or more internaltrans-acting ribozyme. Trans-acting ribozymes act in a target-specificmanner and therefore may act as a toxic agent to a pathogen (such asbacteria) or a selected cell (such as a diseased cell). In accordancewith the present invention, the multi-ribozyme may comprise a) atrans-acting ribozyme flanked by 5′ and 3′ autocatalytically cleavingribozymes or enhanced autocatalytically cleaving ribozymes; b) atrans-acting ribozyme flanked by either a 5′ or 3′ autocatalyticallycleaving ribozyme; or c) multiple transacting ribozymes, flanked by oneor both 5′ and 3′ autocatalytically cleaving ribozymes or enhancedautocatalytically cleaving ribozymes. Multi-ribozymes of the inventionmay also be used to deliver one or more toxic agents to a pathogen cellor tissue. Ribozymes useful in the present invention include thosedescribed in U.S. Pat. No. 5,824,519 and PCT publications No.WO98/24925, WO97/17433, WO98/24925, WO99/67400, which are incorporatedby reference herein in their entirety. In accordance with the presentinvention the multi-transacting ribozymes may be targeted to the samesite on the same RNA, different sites on the same RNA or different RNAs.In accordance with the present invention the multiple toxic agents maybe targeted to the same site on the same target (such as a cellular RNAor protein), different sites on the same target or different targets.For example, in certain embodiments a toxic agent (such as an antisensenucleic acid or nucleic acid encoding a toxic protein) may be engineeredinto a multi-ribozyme in place of a transacting ribozyme, or in additionto a trans-acting ribozyme. In this embodiment, the toxic agent isflanked by a 5′ and/or 3′ autocatalytically cleaving ribozyme.

[0018] The invention additionally provides nucleic acids and expressioncassettes which encode the toxic agent and/or ribozymes of theinvention. These nucleic acids can be used to express the toxic agent(s)and/or ribozyme(s) of the invention at the selected site.

[0019] At the molecular genetic level the coding sequence for a toxicagent, ribozyme, or multi-ribozyme of the invention may be placed underthe control of one or more of the following genetic elements: anaturally occurring strong, intermediate, or weak constitutivelyexpressed or regulated promoter from the targeted microorganism, or anartificially contrived constitutively expressed or regulated promotercontaining either a strong, intermediate or weak consensus sequence thataccords the desired levels of ribozyme and/or toxic agent expression.The present invention relates to promoter elements which arepathogen-specific. The invention relates to promoter elements which areused to achieve pathogen-specific expression of the toxic agents of thepresent invention. The present invention also relates to promoterelements which are tissue-specific. The invention relates to promoterelements which are used to achieve tissue-specific expression of thetoxic agents of the present invention.

[0020] In one embodiment, the nucleic acids comprise a tissue-specificpromoter operably linked to a sequence encoding one or more toxicagent(s). In another embodiment, the nucleic acids comprise apathogen-specific promoter operably linked a sequence encoding one ormore toxic agent(s). In accordance with the present invention, toxicagents of the invention may act on the same or different targets.

[0021] The present invention relates to a toxic agent and/or atrans-acting ribozyme which targets any cellular, viral, bacterial,fungal, or other single cellular or multicellular organism from anyknown taxonomic family, genus, or species. Another embodiment of theinvention relates to a toxic agent which is lethal or toxic to apathogen such as a bacteria, fungus, yeast, diseased cell.

[0022] The targets of the antimicrobial ribozyme therapeutics describedherein are the RNAs of invading or normal flora microorganisms. Thetargets of the antimicrobial toxic agent therapeutics described hereininclude RNAs, proteins, genes and other molecules of invading or normalflora microorganisms. The invention provides the delivery of a series ofribozymes and/or toxic agents directed towards essential, housekeeping,or virulence genes of one or a series of candidate microorganisms.Inactivation of essential proteins and virulence determinants render theinvading microbes inactive or slow their growth, while at the same time,the essential processes of the host are not significantly affected.

[0023] The present invention also relates to the delivery of the toxicagents of the invention to cell or pathogen by abiologic or biologicsystems. In a specific embodiment, a toxic agent of the invention isdelivered to a bacterial cell by a modified bacteriophage capable ofinfecting a pathogenic bacteria. In a further embodiment, bacteriophageare selected for their ability to infect a particular species or generaof bacteria, and are used to deliver a toxic agent for the eradicationof such bacterial species or genera from a host. In a preferredembodiment, the delivery vehicle or nucleic acids native to the deliveryvehicle are modified such that they contain insufficient geneticinformation for the delivery of nucleic acids native to the deliveryvehicle. Thus, the modified delivery vehicle (e.g., virion orbacteriophage) can serve as a molecular vehicle that delivers theribozyme(s) and/or toxic agent(s) of the invention to the target cell orpathogen, but does not deliver replicable nucleic acids native to thedelivery vehicle. Alternatively, an abiologic delivery system (e.g.,liposomes) can be used to package nucleic acid carrying the geneticelements necessary and sufficient for the proper expression of theribozyme(s) and/or toxic agent(s). In one embodiment, delivery of atoxic agent to a pathogen is by use of a bacteriophage or other deliveryvehicle which targets the pathogen of interest. In one embodiment, arecombinant bacteriophage delivers the toxic agent or nucleic acidsencoding the toxic agent to the pathogen.

[0024] The present invention provides compositions of matter which hasresulted from the development of methods and compositions for thedelivery of one or more ribozymes and/or toxic agents directed againstfundamental and essential cellular processes specific to a targetedmicroorganism through an inactivated, altered, virus (virion),bacteriophage, or abiologic delivery vehicles, capable of delivering anucleic acid comprising the toxic agent(s) and/or ribozyme(s) into thetargeted microorganism. The microorganisms may be any virus, nonvirus,bacterium, or lower eukaryotes such as fungi, yeast, parasites,protozoa, or other eukaryotes that may be considered pathogens ofhumans, animals, fish, plants, or other forms of life. Thus, theinvention has important implications in human and veterinary medicine.

[0025] In certain preferred embodiments, a toxic agent of the inventionis used as an antimicrobial therapeutic. A toxic agent may be usedalone, or in combination with one or more other toxic agents. Thus,delivery of a toxic agent to an invading microorganism, kills or renderit less fit. A toxic agent may also be used in combination with one ormore ribozymes. Further, a combination of ribozymes and toxic agents maybe used as an antimicrobial therapeutic.

[0026] The toxic agent approaches of the invention offer advances forantimicrobial therapeutics including but not limited to: (1) the bypassof de novo or built-in drug resistance, which sophisticated microbes mayhave or develop (2) the decreased ability of cells to counteractribozymes or toxic agents delivered into them, (3) the use of broad RNAtargets and non-RNA targets available in microbes that can be attackedin simultaneously (4) the flexibility of custom design of the presentdelivery vehicle can be readily tailored to different families oforganisms or different species of organisms, (5) the ease of assemblyconstruction and manufacture of the modified delivery vehicle, (6) theavailability of a variety of methods of administration of thepharmaceutical preparations of the invention such as topically, or viainjection, inhalation, or ingestion, etc. (7) the ability to lyophilizethe pharmaceutical preparation and thus confer stability to theantimicrobial therapeutic, (8) the reduced immunogenicity of thetherapeutic preparations, and (9) the availability of animal testsystems that enable the evaluation of the ribozymes and/or toxic agentsof the invention. Therefore, the unique delivery approach and anaggressive mechanism for depriving the pathogen essential or importantgene products can achieve the timely defeat of pathogen within theinfected host. Accordingly, the invention has important implication inthe eradication of drug-resistant pathogens.

[0027] The present invention is also directed to a purified preparationof at least one nucleic acid molecule that specifically hybridizes underphysiological conditions to mRNA encoding at least one viral proteinassociated with transformation or plasmid copy number control or whichhybridizes to a viral polyadenylation signal. A further embodiment ofthe present invention is directed to a purified preparation of at leastone nucleic acid molecule wherein said mRNA is E6/E7 mRNA. A stillfurther embodiment of the invention is directed to a purifiedpreparation of at least one nucleic acid molecule wherein said mRNA ispapilloma viral RNA or hepatitis B viral RNA. The present inventionprovides a purified preparation of one or more antisense nucleic acidsthat specifically hybridize under physiological conditions topapillomavirus E6/E7 mRNA or papillomavirus polyadenylation signalwherein said antisense nucleic acids are antisense oligonucleotides,ribozymes or DNAzymes.

[0028] A further embodiment of the present invention is directed to apurified preparation of at least one nucleic acid molecule thatspecifically hybridizes under physiological conditions to a sequenceselected from the group consisting of SEQ ID NOs:AA-BD or a homologoussequence in a related HPV or HBV strain. A homologous sequence in arelated HPV or HBV strain is preferably at least about 70% or 75% or 80%or 85% or 86% or 87% or 88% or 89% or 90% or 91% or 92% or 93% or 94% or95% or 96% or 97% or 98% or 99% identical to a target site identified inthe instant disclosure.

[0029] A further embodiment of the present invention is directed to apurified preparation of at least one nucleic acid molecule as describedabove wherein said nucleic acid molecule is a DNAzyme or an antisenseoligonucleotide or a ribozyme, either alone or in combination.

[0030] A further embodiment of the present invention is directed to apurified preparation of any of the preceding nucleic acid molecules,wherein said nucleic acid molecules are made resistant to nucleasedegradation by any means known in the art.

[0031] In a preferred embodiment, nucleic acid molecules of the presentinvention are modified at their 3′ ends to resist nuclease degradationby inclusion of a 3′-3′ inverted T at the 3′ ends. The modification atthe 3′ ends may be to include a 3′-3′ inverted thymidine, adenine,guanine or cytosine (inverted T, inverted A, inverted G, inverted C,respectively) at the 3′ ends.

[0032] A further embodiment of the present invention is directed topharmaceutical compositions comprising any of the preceding nucleic acidmolecules and a pharmaceutically acceptable carrier. In a still furtherembodiment, any of the pharmaceutical compositions may be formulated ascosmetic formulations and/or formulations for topical administration.The topical formulations may be in the form of ointments, salves, gels,creams or lotions. Any of the pharmaceutical compositions may beformulated such that the nucleic acid molecules are formulated into aliposome preparation or a lipid preparation. In a further embodiment,the liposome is capable of tissue-specific uptake in the liver. In astill further embodiment said liposome is modified using asialofetuin orone or more sugars.

[0033] A still further embodiment is directed to pharmaceuticalcompositions wherein the nucleic acid molecules of the present inventionare formulated in amounts sufficient to produce cytotoxic or cytostaticeffects in cells infected with papillomavirus or heptatis B virus. Thecells may also be transformed by a papillomavirus or transformed by ahepatitis B virus.

[0034] A further embodiment of the present invention is directed tomethods of treating papillomavirus-induced conditions orhepatitis-induced conditions comprising administering to a subject anyof the preceding pharmaceutical compositions. In a further embodimentsaid papillomavirus-induced condition is selected from the groupconsisting of warts (e.g., warts of the hands, feet, larynx, and/or flatcervical warts), cervical carcinoma, laryngeal papilloma, condylomataacuminata, epidermodysplasia verruciformis, cervical intraepithelialneoplasia, or any other infection involving a papillomavirus. Themethods of the instant invention include the treatment of epithelialcells such as squamous epithelia, cutaneous epithelia and mucosalepithelia, or any other cell infected or which may become infected witha papillomavirus.

[0035] A further embodiment of the present invention is directed tomethods of administration of any of the preceding pharmaceuticalcompositions comprising topical application. The administration may beto the cervix or the epidermis. In a further embodiment saidadministration is to epithelial cells such as squamous epithelia,cutaneous epithelia and mucosal epithelia.

[0036] The instant invention is also directed to methods of treatingpapillomavirus-induced conditions comprising: administering to asubject, by topical application to cells infected with saidpapillomavirus, one or more of the antisense oligonucleotides, DNAzymesor ribozymes described herein. The method of treatingpapillomavirus-induced conditions includes cervical application ordermal or epidermal application.

[0037] In a still further embodiment of the invention, the methods oftreating papillomavirus include treatments of conditions induced byhuman papillomavirus (HPV).

[0038] In a further embodiment, the pharmaceutical compositions of theantisense nucleic acids of the invention may be cosmetic formulations.

[0039] The pharmaceutical compositions may be formulated such that thenucleic acids of the invention are present in amounts sufficient toproduce a cytotoxic or cytostatic effect in cells infected with a wartvirus or viruses. In a specific embodiment, the wart virus is apapillomavirus. In a still further embodiment, the cells are transformedby a papillomavirus.

[0040] In a preferred embodiment, pharmaceutical compositions ofDNAzymes, antisense oligonucleotides or ribozymes of the instantinvention are formulated for topical administration. Such formulationsinclude ointments, salves, gels, creams, lotions or suppositories.

[0041] In a still further embodiment of the instant invention, thepharmaceutical composition of the nucleic acids of the present inventionmaybe formulated into a liposome preparation or a lipid preparation.

[0042] A pharmaceutical composition may comprise one or more nucleicacids of the invention selected from the group consisting of antisenseoligonucleotides, DNAzymes or ribozymes, wherein said nucleic acidsspecifically hybridize under physiological conditions to HPV E6/E7 mRNAor an HPV polyadenylation signal, and/or wherein said antisenseoligonucleotides or DNAzymes have a 3′-3′ inverted thymidine at their 3′ends, and/or wherein said pharmaceutical composition is formulated fortopical application as an ointment, salve, gel, cream or lotion.

[0043] In another preferred embodiment, a purified preparation of one ormore nucleic acids of the invention that specifically hybridize underphysiological conditions to hepatitis B viral (HBV) RNA are provided,wherein said one or more nucleic acids are antisense oligonucleotides,DNAzymes or ribozymes.

4. BRIEF DESCRIPTION OF THE FIGURES

[0044]FIG. 1A Diagram depicts the components of the lacI-regulated broadspectrum promoter.

[0045]FIG. 1B The sequence of the LEASHI promoter (SEQ ID NO:1).

[0046]FIG. 1C The sequence of a modified rrnB promoter (SEQ ID NO:2).

[0047]FIG. 1D The sequence of the Anr promoter (SEQ ID NO:3).

[0048]FIG. 1E The sequence of the Proc promoter (SEQ ID NO:4).

[0049]FIG. 1F The sequence of the Arc promoter (SEQ ID NO:5).

[0050]FIG. 1G The sequence of the TSST-1 promoter (SEQ ID NO:6).

[0051]FIG. 2 Diagram of a β-lactamase reporter plasmid.

[0052]FIG. 3A-B Expression vectors for cloning Toxic Agents.

[0053]FIG. 4 Assay for lethality of Toxic Agents

[0054]FIG. 5 Growth of E. coli harboring a doc expression plasmid.

[0055]FIG. 6A-B Structure of a Transfer Plasmid.

[0056]FIG. 7 Delivery Efficiency of the Transfer Plasmid by the P1bacteriophage vehicle to E. coli.

[0057]FIG. 8 Scheme for generation of the P1 pac site knockout.

[0058]FIG. 9 Identification and confirmation of the P1 pac site knockoutby PCR screening.

[0059]FIG. 10 Diagram of the pacABC Complementing plasmid.

[0060]FIG. 11 Recombination between the P1 pac mutant and the pacABCComplementing plasmid.

[0061]FIG. 12 Sequence of the minimal P1 pac site (SEQ IS NO:7).

[0062]FIG. 13 Immunogenicity of replicating phage in mice.

[0063]FIG. 14 Comparison of original and long-circulating P1 phagepersistence in vivo.

[0064]FIG. 15 Treatment of P. aeruginosa (PA01) infections inembryonated hen eggs.

[0065]FIG. 16 In vitro killing of E. coli EC-4 bacterial cells.

[0066]FIG. 17 Treatment of E. coli EC-4 infection in embryonated heneggs.

[0067]FIG. 18 Sequence of the DicF1 molecule (SEQ ID NO:8).

[0068]FIG. 19 Diagram and nucleotide sequence of the pClip ribozymecassette.

[0069]FIG. 20 Diagram and nucleotide sequence of the pChop ribozymecassette.

[0070]FIG. 21 Schematic diagram of the pSnip ribozyme cassette. pSnipincludes sequences of the pClip triple ribozyme cassette, catalytic coretargeted ribozymes comprising two linked trans-acting ribozymes, andsequences from the pChop triple ribozyme cassette.

[0071]FIG. 22A A schematic of DNA encoding the ribozyme used in themolecular sequence of events in ribozyme maturation and action.

[0072]FIG. 22B The primary RNA transcript. Autocatalytic cleavage takesplace upon completion of transcription.

[0073]FIG. 22C The release of the trans-acting ribozyme. As a directresult of cleavage of the two cis-acting ribozymes, the internalribozyme containing a reverse and complementary sequence to the mRNAtarget is released.

[0074]FIG. 22D The sequence specific hybridization of the ribozyme. Theinternal or trans-acting ribozymes comprise two trans-acting ribozymeslinked by a short nucleotide “spacer”. Each of the two trans-actingribozymes contain a sequence that is reverse complementary to thetargeted message of the same or at different sites. The ribozyme issynthesized at a concentration sufficient to locate and hybridize to allor substantially all targeted transcripts.

[0075]FIG. 22E The trans-catalytic cleavage. Upon hybridization of theinternal trans-acting ribozyme to the targeted mRNA transcript, theinternal ribozyme achieves a catalytic topology and cleaves the targetedmessage. Upon cleavage the trans-acting ribozyme is released and itsactivity and function are recycled.

[0076]FIG. 23 In vitro cleavage analyses of HBV-targeted Rz. Thelocations of the 6 Rz tested are as shown in FIG. 7 of priorityprovisional application No. 60/251,810 filed Dec. 7, 2000, incorporatedby reference herein. Individual Rz were constructed and transcribed invitro, and mixed with [³²P]-labeled HBV target RNA (917 nt). Incubationswere for 30 min at 37 C in 20 mM Tris-HCl (pH 7.4), 5 mM MgCl₂.Following cleavage, products were separated by denaturing PAGE, andresults were quantitated using a Phosphor-Imager. The size of thecleavage products is shown to the right, and the concentration of Rz (40mM or 200 mM) is shown at the top.

[0077]FIG. 24 Cleavage analyses of HBV-targeted Rz. Rz and HBV-targetRNA were mixed at various ratios (40:1, 40:10,or 40:100,as shown at thetop), and incubated as described for various periods (20 sec, 40 sec, 1min, 3 min, 10 min, 30 min, or 2 h for each ratio, shown from left toright, respectively). After incubation, products were analyzed bydenaturing PAGE and quantitated using a Phosphor-Imager.

[0078]FIG. 25 Effects of topical application of DNAzymes on papillomagrowth in cottontail rabbits. DNAzymes targeted to Shope Papilloma VirusmRNA sites were applied alone (Group L1, Group L2, Group L3) or incombination (Group L1/L2/L3). Results showed that the combinationtherapy was effective in reducing papilloma volume. Catalyticallyinactive DNAzyme (Group mL2) was ineffective in reducing papillomavolume.

5. DETAILED DESCRIPTION OF THE INVENTION

[0079] The present invention provides toxic agent(s) and/or ribozyme(s)and their use in a tissue-specific, target-specific, orpathogen-specific manner for the treatment of disorders and diseaserelated to bacterial, parasitic or viral infections or to cellularproliferation, and cancers. The ribozymes and/or toxic agents of thepresent invention may be engineered to target one or more specific RNAscontained in a specific cell or tissue in the host. The ribozymes of thepresent invention may also be engineered to target one or more specificRNAs encoded by a specific pathogen, virus, or microbial agent. Thetoxic agents of the present invention may also be engineered to targetone or more specific RNAs, proteins, or molecules of a specificpathogen, virus, or microbial agent.

[0080] The present invention also provides toxic agents which are lethalor toxic to a selected pathogen. In one embodiment of the invention, thetoxic agents of the invention comprise toxic proteins which causelethality to a pathogen or selected cell (e.g., a diseased cell) orwhich render the pathogen or selected cell less fit. In one embodiment,such toxic proteins of the invention are lethal when overexpressed in apathogen or selected cell. In other embodiments, a toxic protein is anexogenous protein that is toxic when expressed in a pathogen or selectedcell. A toxic protein of the invention may further be engineered to haveincreased toxicity. For example, many methods are known in the art forintroducing mutations, deletion, insertions etc. into a known sequence.Thus, optimization of a toxic protein is provided. The invention alsoprovides methods for inhibiting the toxicity of a toxic protein, so thatthe toxic protein may be produced or manufactured in a producing cell.Inhibiting the toxicity may be performed by any methods known in theart, for example, the toxic protein may be expressed from an induciblepromoter which allows expression to be turned on/off under appropriateconditions. A toxic protein may be expressed in a cell without causinglethality in the cell by overexpressing an antidote protein in the samecell. Other methods will be apparent to one skilled in the art and arewithin the scope of the invention.

[0081] The present invention provides toxic agents and methods forspecifically targeting toxic agents to bacteria or bacteria-infectedcells or other pathogens. Toxic agents of the present invention aredirected to one or more targets and thus can be used alone or incombination to eradicate bacteria. Specifically, the invention providesthe delivery of one or more toxic proteins, antisense RNAs,multi-ribozymes, or nucleic acids encoding the same, or a combinationthereof, to a cell, tissue, or subject containing an infectious bacteriaor pathogen in order to eradicate such bacteria or pathogen.

[0082] The present invention further encompasses the use of the toxicagents and/or ribozymes of the present invention as therapeutics andpharmaceutical compositions. In specific embodiments of the invention,the toxic agents of the invention are useful to treat microbialinfections associated with severe burns, cystic fibrosis, cancer, orother immunocompromising conditions.

[0083] The present invention further encompasses the use of the toxicagents and/or ribozymes of the present invention for research andscreening purposes. In one embodiment of the present invention, theribozymes and/or toxic agents may be used to screen for viral,microbial, prokaryotic, or eukaryotic gene products or molecules to betargeted in order to effectively inhibit the selected virus or microbialagent or selected cell.

[0084] 5.1. Pathogen-Specific and Tissue-Specific Toxic Agents

[0085] The invention provides specific nucleic acids which act as orencode toxic agents and are therefore useful as antimicrobial agents. Avariety of toxic agents are within the scope of the invention. Forclarity, the toxic agents of the invention are described herein below inseveral sub-types. The toxic agents of the invention include but are notlimited to antisense nucleic acids, toxic gene products, sense nucleicacids.

[0086] 5.1.1. Toxic Gene Products

[0087] The present invention relates to the use of toxic gene productsor toxic proteins as toxic agents for the treatment of disorders anddisease related to bacterial, parasitic, fungal, or viral infections orto cellular proliferation, and cancers, or to diseased cells. A toxicgene product of the invention is any gene product (such as DNA, RNA orprotein), which is toxic to a pathogen or selected cell (such as adiseased cell). Such toxic gene products may be naturally occurring(endogenous), or may be non-naturally occurring (exogenous) in thetarget pathogen or selected cell. A toxic agent of the invention may bea chromosomally encoded, plasmid encoded, pathogen encoded, synthetic,or encoded in any other nucleic acid or nucleotide sequence. The presentinvention provides toxic agents which are endogenous toxic gene productsthat are expressed in a pathogen or selected cell which kill or renderthe pathogen or selected cell less fit. The present invention alsoprovides toxic agents which are exogenous toxic gene products that areintroduced into or expressed in a pathogen or selected cell which killor render the pathogen or selected cell less fit. A pathogen or selectedcell which is less fit is one which is weakened, or which is moresusceptible to chemical treatment (such as drugs, toxins,pharmaceuticals, mutagens, solvents, etc.), or which is more susceptibleto physical stress (such as temperature), or which is more susceptibleto genetic alterations (such as by radiation or UV), or is moresusceptible to environmental changes (such as available nutrients).

[0088] In several embodiments, the present invention provides the use ofa plasmid addiction system protein as a toxic agent when expressed inbacteria or a selected cell. For example, in certain types ofbacteriophage, the lysogenic (dormant) pathway is manifested by abacterial cell maintaining only a single copy of the bacteriophage DNAin the form of a plasmid. In order to assure that both daughterbacterial cells receive a copy of the plasmid, a “plasmid addictionsystem” or “post-segregation system” is used by the cells which ensuresthat only bacterial cells which receive a copy of the plasmid willsurvive.

[0089] In one embodiment of the invention, a post-segregation system orplasmid addiction system toxin, is used as a toxic agent to a pathogen(such as bacteria) by overexpression of the toxin. Such overexpressionof the toxin uncouples the toxin and the antidote, leading to toxicity,and preferably lethality, in the cell containing the overexpressed toxicagent.

[0090] For example, in one embodiment, the invention provides toxicagents which specifically target gene products essential for thesurvival or life cycle of a pathogen (such as replication, packagingetc). In one embodiment, the present invention provides naturallyoccurring addiction system toxins which have been modified to beexpressed in the absence of the addiction system antidote. In anotherembodiment, the present invention provides naturally occurring addictionsystem toxins which have been modified to be expressed at higher levelsthan the addiction system antidote. In one example, an addiction systemtoxin (e.g., doc, chpBK, kicB, or gef) is used as a toxic agent and isuncoupled from its antidote. In another embodiment of the invention, achromosomally encoded toxic gene product (such as chpBK, kicB, or gef)is used as a toxic agent to a pathogen by overexpression of the toxicgene product.

[0091] In certain embodiments, toxic agents include but are not limitedto Shiga-like toxins of E. coli, cholera toxin of Vibrio cholerae, andcytotoxins of P. aeruginosa. For example, phage K139 confers to V.cholera a gene product that enhances enzymatic activity of choleratoxin. Such toxins are within the scope of the invention and may be usedas a toxic agent in association with the methods and compositions of theinvention. In certain embodiments of the invention, the baceriocidaltoxic agent is derived from a bacterium including but not limited toStaphylococcus aureus, Enterococcus faecalis, or Pseudomonas aeruginosa.

[0092] In another embodiment, the antidote of a toxin is the target of atrans-acting ribozyme or toxic agent of the invention. Thus, when theantidote is inactivated by the trans-acting ribozyme or toxic agent, thetoxin is no longer neutralized or inactivated by the antidote, thusleading to toxicity, and preferably lethality to the pathogen.

[0093] In yet another embodiment, when the antidote is itself anantisense RNA, a sense RNA may be synthesized as a toxic agent anddelivered to inactivate the antisense antidote. Thus, when the antidoteis inactivated by the sense RNA, the antidote is no longer available toinactivate the toxin, thus leading to toxicity, and preferablylethality.

[0094] One example of an addiction system toxin that may be used inconnection with the invention is doc (death on curing; Lehnherr H, etal., 1993, J. Mol. Biol. 233:414-28). The protein encoded by doc islethal or toxic in both Gram-negative and Gram-positive organisms (e.g.,E. coli, P. aeruginosa, Staphylococcus aureus, and Enterococcusfaecalis). doc acts as a bacterial cell toxin to which Phd (preventionof host death) is the antidote. Accordingly, the invention provides forplasmids expressing doc which can be delivered to a bacterial pathogenin order to render the pathogen less fit, and preferably eradicate thepathogen. A particular advantage of doc is that doc has little to notoxicity to eukaryotic cells, and thus may be administered safely to aeukaryotic host.

[0095] Specific examples of addiction system toxins or chromosomallyencoded toxins, or other toxic agents which may be used in connectionwith the invention include but are not limited to ccdB, kid, perK, parE,doc, higB, chpAK, chpBK, kicB, hoc, srnB′, flmA, pmda, relF, gef, kilA,kilB, kilC, kilE, traL, traE, sigB, hok, pemK, lysostaphin, and kikA.Examples of antidotes which may be used as in the methods of theinvention include but are not limited to ccdA, kis, pemI, parD, phd,higA, chpAI, chpBI, kicA, soc, srnC, flmB, pndB, sof korA, korB, korC,korD, korE, and korF. Thus, the invention herein provides a method ofusing a an addiction system toxin (such as doc) or other toxic protein,as a toxic agent of the invention. The invention also provides methodsfor inhibiting or inactivating antidotes of a toxin. The inventionfurther provide co-expression of a toxin and its corresponding antidotefor manufacturing purposes.

[0096] In certain specific embodiments, the invention provides toxicagents chpBK, kicB, and gef. Each of the proteins of kicB, or gef arelethal in E. coli but not in P. aeruginosa. Accordingly, the inventionprovides for the use of kicB or gef in the eradication or treatment ofbacterial infections of E. coli. In one embodiment, kicB or gef encodingnucleic acids are delivered by a to the E. coli by a P1 bacteriophage ofthe invention containing a transfer plasmid, said transfer plasmidencoding the kicB or gef toxic agents (or both).

[0097] In another specific embodiment of the invention, the chpBKprotein is a toxic agent of the invention and is lethal to E. coli andtoxic or lethal in P. aeruginosa. Accordingly, the invention providesfor the use of chpBK in the eradication or treatment of infections of E.coli or P. aeruginosa. In one embodiment, chpBK nucleic acids aredelivered by a to E. coli or P. aeruginosa by a P1 bacteriophage of theinvention, containing a transfer plasmid, said transfer plasmid encodingthe chpBK toxic agent. The antidote protein that antagonizes chpBKfunction is called ChpBl. Accordingly, the invention provides, for theco-expression of the antidote ChpBl and chpBK for manufacturingpurposes.

[0098] In several embodiments of the invention, the toxic gene, such asdoc, chpBK, kicB, or gef is placed under the control of an induciblepromoter and is uncoupled from the antidote. In one embodiment, thepromoter is the P1 lytic promoter P53. In a preferred embodiment, thepromoter is the LEASHI promoter. In a preferred embodiment, for thetreatment of P. aeruginosa infections, the invention provides P.aeruginosa specific promoters, anr, arc or proc.

[0099] In other specific embodiments of the invention, a consensusribosome binding site (GGAGGTGXXXXATG, wherein X is any nucleotide) maybe inserted immediately upstream of the nucleic acids encoding the toxicagent and leads to increased expression of the toxic agent. The providesfor the use of a combination of a promoter and a ribosome entry site(s)to modulate expression of a toxic agent or ribozyme.

[0100] It is also within the scope of the invention that more that onetoxic agent may be used to eradicate or treat an infection. For example,it is contemplated that two or more toxic agents may be engineered intoa single transfer plasmid for delivery by a bacteriophage. Suchbacteriophage could serve to deliver nucleic acids encoding multipletoxic agents to target bacteria. Alternatively, two or more transferplasmids may be carried by a single bacteriophage, wherein each transferplasmid encodes different toxic agents. In this embodiment, when morethan one transfer plasmid is used, such plasmids are designed such thatthe two or more plasmids are non-recombinigenic. Such methods ofengineering non-recombinigenic sequences are known in the art.Additionally, in this embodiment, the two or more engineered plasmidswill preferably have different origins of replication. In this manner,the bacteriophage serves to deliver nucleic acids encoding multipletoxic agents. In yet a third alternative, bacteriophage may be designedto carry multiple toxic agents on multiple transfer plasmids. When twoor more toxic agents are encoded within a single bacteriophage, thenucleic acids encoding such toxic agents may be operably linked to thesame promoter, or different promoters (e.g., see sections 5.4 and 5.4.1herein).

[0101] 5.1.2. Antisense

[0102] The invention provides specific nucleic acids which act as toxicagents and are therefore useful as antimicrobial agents. The inventionprovides antisense RNA molecules which target an RNA of a pathogen orselected cell. Target RNAs of the invention may be pathogen-specificRNAs, tissue-specific cellular RNAs, or disease-specific RNAs. Theinvention also provides modified and enhanced antisense nucleic acidswhich target pathogen-specific RNAs, tissue-specific cellular RNAs, ordisease-specific RNAs.

[0103] The proposed target of the toxic antisense molecule of theinvention is the RNA of a gene which plays a critical role in thesurvival of the pathogen, or which is essential to the pathogen's lifecycle. The present invention also encompasses modifications to naturallyoccurring antisense molecules which modulate the expression of anessential gene product of a pathogen. For example, as described below,one proposed target of an antisense of the invention is the ftsZ genewhose gene product plays a critical role in the initiation of celldivision of E. coli.

[0104] In another embodiment, the toxic agents of the invention compriseantisense molecules designed to have enhanced inhibition of target RNAs.The toxic agents which comprise antisense molecules of the invention areengineered to more specifically bind target RNAs in that the sequencesof such toxic antisense molecules are designed to have increasedcomplementarity to a target sequence such as an essential RNA of apathogen or selected cell. Such toxic antisense molecules are thereforemore specific to their targets and hence, have increased efficacy. Theinvention provides antisense toxic agents and ribozymes which are alsomodified with a hairpin structure to create a more stable molecule. Theantisense toxic agents of the invention may also be expressed to a highlevel in a target pathogen or cell by any method known or cell by anymethod known in the art. For example, an antisense toxic agent may beexpressed in trans from a multi-copy expression plasmid using a strongregulatable promoter. The antisense toxic agent may also be operablylinked to a tissue-specific or pathogen-specific promoter such that theantisense molecule is only expressed in a pathogen or cell which usesthe same promoter.

[0105] Specifically, the invention provides antisense RNAs which targetessential nucleotide sequences, such as DicF1 or a DicF1-like antisensemolecule that specifically target a nucleotide sequence which encodes aprotein essential for replication or survival. Further, the inventionprovides modified antisense structures with increased stability to actas lethal agents when expressed in bacteria. The invention also providestoxic sense molecules designed to target essential antisense molecules.

[0106] In another embodiment of the invention the toxic agents comprisesense RNA molecules targeted to antisense RNAs which are required forthe survival of the pathogen or cell. For example, an antidote of atoxic protein (such as an addiction system toxin) may be in the form ofan antisense molecule which regulates the expression of the toxin. Suchan antisense antidote allows the pathogen or cell to survive in thepresence of such toxin. The invention provides inhibition of theantisense antidote by a toxic agent in the form of a sense RNA molecule.

[0107] In certain embodiments a combination of two or more toxicmolecules may be delivered to a pathogen (such as E. coli, P.aeruginosa, etc.) in order to cause lethality. In this embodiment, thetoxic antisense may be directed to the same target, or differenttargets. When different targets of a pathogen or cell are targeted, suchtargets may be involved in the same biological pathway within thepathogen or different biological pathways.

[0108] In a specific embodiment, the antisense sequence is based on DicF(Bouche F, et al., 1989, Mol Microbiol. 3:991-4). Such modified DicFsequence is referred to as DicF1 (SEQ ID NO:8). Naturally occurring DicFis part of an intercistronic region that when expressed in Escherichiacoli causes inhibition of cell division. This inhibition does notrequire the translation of DicF mRNA into protein, instead, DicF RNAexerts its inhibitory effect as an antisense molecule.

[0109] The proposed target of DicF is the ftsZ gene whose gene productplays a critical role in the initiation of cell division of E. coli.Temperature sensitive mutations of the ftsZ gene indicate that it isessential for viability of E. coli. Without limitation as to mechanism,DicF RNA is believed to bind specifically to the 5′ untranslated regionof ftsZ mRNA, thereby inhibiting ftsZ protein expression. Cells lackingthe ftsZ protein are unable to divide and ultimately die. DicF homologshave been identified in a variety of other bacteria although it is notknown whether they exert a similar function.

[0110] The present invention provides for modified DicF nucleic acids,called DicF1 or DicF1-like RNAs, which are used as antimicrobial agents,or toxic agents of the invention. DicF1 RNA is a superior antisensemolecule as compared to the endogenous DicF RNA. It has been modified byincreasing its complementarity to the ftsZ 5′ untranslated mRNA. It istherefore more specific to its target and hence, has increased efficacy.An auto hairpin structure has further been enhanced to create a morestable molecule. The invention also provides modifications of othernaturally occurring antisense molecules, such as nucleotide sequenceswhich have similar functions as DicF in modulating the expression ofgene products essential to the pathogen's life cycle or survival. Suchnucleic acid is referred to as a DicF1-like nucleic acid. In contrast tothe endogenous DicF, the DicF1 or a DicF1-like nucleic acid of theinvention may be expressed in trans from a multi-copy expressionplasmid. Further, the DicF1 or DicF1-like nucleic acids may be operablylinked to a variety of promoters that may be used to control thestrength, timing, or distribution of such expression. DicF1 or aDicF1-like nucleic acid may also be expressed in trans from a ribozymecassette. The combination of these features results in DicF1 orDicF1-like nucleic acid being an effective antimicrobial agent against apathogen (such as E. coli). In other embodiments, modifications to thesequence of an antisense of the invention allows targeting against avariety of other bacteria. In other embodiments, modifications to thesequence of an antisense of the invention allows targeting in apathogen-specific manner. The invention also provides DicF1-like nucleicacids which may be used as toxic agents in bacteria, bacteria-infectedcells, or other pathogens which have complementary RNA targets.

[0111] 5.1.3. Antisense Oligonucleotides

[0112] Antisense oligonucleotides that hybridize or anneal underphysiological conditions to at least a portion of a target sequence arealso provided for use in the compositions and methods of the invention.Such oligonucleotides are typically short in length (e.g. any number ofcontiguous nucleotides or analogues thereof from about six to aboutfifty) and can be delivered to cells by any methods known in the art(see e.g., exemplary methods of administration set forth in section 5.9below). Such antisense oligonucleotides include, but are not limited to,polydeoxynucleotides containing 2′-deoxy-D-ribose, polyribonucleotidescontaining D-ribose, any other type of polynucleotide which is anN-glycoside of a purine or pyrimidine base, or other polymers containingnon-nucleotide backbones (e.g., peptide nucleic acids or PNA, see below,and any other synthetic sequence-specific nucleic-acid-like polymerwhich is commercially available) or nonstandard linkages, providing thatthe polymers contain nucleotides in a configuration which allows forbase pairing and base stacking such as is found in DNA and RNA. They mayinclude double- and single-stranded DNA, as well as double- andsingle-stranded RNA and DNA:RNA hybrids, as well as all known types ofmodifications, for example, labels, “caps”, methylation, substitution ofone or more natural nucleotides with one or more analogues. Additionalknown modifications include internucleotide modifications such as, forexample, various uncharged linkages (e.g., methyl phosphonates,phosphorotriesters, phosphoramidates, carbamates, etc.), chargedlinkages or sulfur-containing linkages (e.g., phosphorothioates,phosphorodithioates, etc.), pendant moieties (such as, for example, onproteins including nucleases, nuclease inhibitors, toxins, antibodies,signal peptides, poly-L-lysine, etc.) and saccharides (e.g.,monosaccharides, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, boron, oxidative metals,etc.), alkylating agents, and modified linkages (e.g., alpha anomericnucleic acids, etc.).

[0113] Peptide nucleic acid (PNA) is just one example of a nucleic acidderivative or analogue well known in the art which may be used in thecompositions and methods of the present invention. PNA was firstdescribed in 1991 by Nielsen et al. (1991, Science 254, 1497-1500) andhas been described as a nucleic acid mimetic with a neutral peptide-likebackbone instead of a negatively-charged sugar-phosphate backbone.However, the same nitrogenous bases (i.e. adenine, guanine, cytosine andthymine) are used in PNA as found in DNA and RNA, and PNA undergoesWatson-Crick base pairing with DNA and RNA. PNA is not generallyrecognized as a substrate for DNA polymerases, nucleic acid bindingproteins, or other enzymes, including proteases and nucleases, althoughsome exceptions exist (see e.g. Lutz et al., 1997, J. Am. Chem. Soc.119, 3177-3178). The chemical structure of PNA consists of repeatingunits of N-(2-aminoethyl)-glycine linked by amide bonds. The nitrogenousbases are attached to this neutral backbone by methylene carbonyllinkages. Unlike the natural nucleic acid backbone, no deoxyribose,ribose or phosphate groups are present. As a result, PNA binding to atarget nucleic acid is stronger than with conventional nucleic acids,and the binding is virtually independent of salt concentration.Quantitatively, this is reflected by a high thermal stability ofduplexes containing PNA.

[0114] PNA may be synthesized by methods well known in the art usingchemistries similar to those used for synthesis of nucleic acids andpeptides. The PNA monomers used in such syntheses are hybrids ofnucleosides and amino acids. The neutral backbone of a PNA oligomerresults in several unique properties. Its properties relative to naturalnucleic acids include: (a) higher affinity; (b) faster hybridization;and (c) relative independence of hybridization from salt concentration.In general, a given PNA duplexed with a natural nucleic acid having oneor more mismatches will result in a greater change in meltingtemperature (i.e. ΔT_(m)). PNA products, services, and technical supportare commercially available from PerSeptive Biosystems, Inc., a divisionof Applied Biosystems (www.pbio.com). For example, PNA may besynthesized using kits or custom PNA synthesis may be ordered. PNAoligomers may also be manually synthesized using either Fmoc or t-Bocbased monomers and standard peptide chemistry protocols. Standardpeptide purification conditions may be used to purify PNA followingsynthesis.

[0115] A variety of extensive PNA reviews have been published, each ofwhich is incorporated by reference herein (see e.g., Nielsen et al.,1992, In Antisense Research and Applications, Crooke and Lebleu, eds.,CRC Press, pp. 363-372; Nielsen et al., 1993, Anti-Cancer Drug Design 8,53-63; Buchardt et al, 1993, TIB TECH 11, 384-386; Nielsen et al., 1994,Bioconjugate Chem. 5, 3-7; Nielsen et al., 1996, In AntisenseTherapeutics Vol. 4 (ed. Trainor, ed., SECOM Science Publishers B. V.,Leiden, pp. 76-84; Nielsen, 1995, Ann. Rev. Biophys. Biomol. Struct. 24,167-183; Hyrup and Nielsen, 1996, Bioorg. Med. Chem. 4, 5-23; Mesmaekeret al., 1995, Curr. Opin. Struct. Biol. 5, 343-355; Dueholm and Nielsen,1997, New J. Chem. 21, 19-31; Knudsen and Nielsen, 1997, Anti-CancerDrug 8, 113-118; Nielsen, 1997, Chem. Eur. J. 3, 505-508; Corey, 1997,TIB TECH 15, 224-229; Nielsen and Orum, 1995, In Molecular Biology:Current Innovations and Future Trends, Part 2, Horizon Scientific Press,pp. 73-89; Nielsen and Haaima, 1997, Chem. Soc. Rev., 73-78; Ørum etal., 1997, In Nucleic Acid Amplification Technologies: Application toDisease Diagnostics, Lee et al., eds., BioTechniques Books Div., EatonPublishing, pp. 29-48).

[0116] 5.1.4. Dnazymes

[0117] DNAzymes have been described in various publications, includingU.S. Pat. No. 6,159,714 to Usman et al., International Publication WO00/09673 to Sun et al. and U.S. Pat. No. 5,807,718 to Joyce et al., allof which are hereby incorporated by reference in their entirety. As theterm is used in this application, DNAzymes (also known in the art ascatalytic DNA enzymes, or Deoxyribozymes) are chimeric DNA, ornon-RNA-containing DNA molecules having an enzymatic activity which isable to cleave (preferably, cleave repeatedly) separate target RNAmolecules in a nucleotide base sequence specific manner.

[0118] DNAzymes act by first binding to a target RNA. Such bindingoccurs through and with the DNA-binding portion of a DNAzyme, which isheld in close proximity to the RNA substrate. The catalytic sequences ofthe DNAzyme then cleave the target RNA. Thus, the DNAzyme firstrecognizes and then binds a target RNA through complementarybase-pairing, and once bound to the correct site, acts enzymatically tocut the target RNA. Strategic cleavage of such a target RNA will destroyits ability to direct synthesis of an encoded protein. After a DNAzymehas bound and cleaved its RNA target, the DNAzyme is released from thatRNA and is free to complex with another target. Thus, a single DNAzymemolecule can bind and cleave new targets repeatedly.

[0119] Generally then, a DNAzyme is a DNA molecule that hascomplementarity in a substrate-binding region to a specified gene targetsequence, and also is able to cause specific cleavage of the RNA targetsequence. That is, the DNAzyme molecule is able to intermolecularlycleave RNA and thereby inactivate a target RNA molecule. Thecomplementarity sequences of a given DNAzyme function to allowsufficient hybridization of the DNAzyme to its target RNA to allow thecleavage to occur. One hundred percent complementarity is preferred, butcomplementarity as low as 50-75% may also be useful in this invention.In particular, mismatches may be tolerated within the target recognitionsite that are not adjacent to the cut region.

[0120] The catalytic core of a DNAzyme may be selected from any of anumber of possible sequences that have enzymatic activity against a RNAsubstrate. In one embodiment, the catalytic core sequence is5′-GGCTAGCTACAACGA-3′ (SEQ ID NO ). Other catalytic core sequences aredisclosed, for example, in U.S. Pat. No. 5,807,718 to Joyce et al.

[0121] 5.1.5. Antisense Oligonucleotides, Dnazymes, and AnaloguesModified at Their 3′ and/or 5′ Termini

[0122] Antisense oligonucleotides modified at their 3′ and/or 5′ endshave been described, for example in U.S. Pat. No. 5,750,669 to Rosch etal., which is hereby incorporated by reference in its entirety. Thecharacteristic structural modification of these oligonucleotides is thatthe internucleotide linkage at the 3′ end is altered, that is to say, a3′-3′ linkage exists in place of the biological 3′-5′ linkage. Thisminimal structural modification suffices to stabilize such compoundsagainst nuclease degradation. Inverted bases such as thymidine,cystosine, guanosine or adenine may be used, although other bases arepossible. This slight structural modification results in a hybridizationbehavior which is almost identical to that of unmodifiedoligonucleotides. This modification also results in these compoundsbeing generally utilizable as inhibitors of gene expression. TheDNAzymes of the present invention may also have the above- disclosedmodifications at their 3′ ends.

[0123] 5.1.6. Nucleic Acid Homology

[0124] Homology or identity at the nucleotide or amino acid sequencelevel may be determined by any method known to the skilled artisanincluding BLAST (Basic Local Alignment Search Tool) analysis using thealgorithm employed by the programs blastp, blastn, blastx, tblastn andtblastx (Karlin et al., 1990, Proc. Natl. Acad. Sci. USA 87, 2264-2268and Altschul, 1993, J. Mol. Evol. 36, 290-300, fully incorporated byreference) which are tailored for sequence similarity searching. Theapproach used by the BLAST program is to first consider similar segmentsbetween a query sequence and a database sequence, then to evaluate thestatistical significance of all matches that are identified, and finallyto summarize only those matches which satisfy a preselected threshold ofsignificance. For a discussion of basic issues in similarity searchingof sequence databases, see Altschul et al., 1994, Nature Genetics 6,119-129) which is fully incorporated by reference. For purposes of thepresent application, search parameters for histogram, descriptions,alignments, expect (i.e., the statistical significance threshold forreporting matches against database sequences), cutoff, matrix and filtermay be used at the default settings. The default scoring matrix used byblastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoffetal., 1992, Proc. Natl. Acad. Sci. USA 89, 10915-10919, fullyincorporated by reference). For blastn, the scoring matrix is set by theratios of M (i.e., the reward score for a pair of matching residues) toN (i.e., the penalty score for mismatching residues), wherein thedefault values for M and N are 5 and −4,respectively. Four blastnparameters were adjusted as follows: Q=10 (gap creation penalty); R=10(gap extension penalty); wink=1 (generates word hits at every wink^(th)position along the query); and gapw=16 (sets the window width withinwhich gapped alignments are generated). The equivalent Blastp parametersettings were Q=9; R=2; wink=1; and gapw=32. A Bestfit comparisonbetween sequences, available in the GCG package version 10.0, uses DNAparameters GAP=50 (gap creation penalty) and LEN=3 (gap extensionpenalty) and the equivalent settings in protein comparisons are GAP=8and LEN=2. A homologous sequence in a related HPV or HBV strain is asequence that is at least about 70% identical to a target site of theinstant disclosure. A homologous sequence in a related HPV or HBV strainis, more preferably, at least about 75% or 80% or 85% or 86% or 87% or88% or 89% or 90% or 91% or 92% or 93% or 94% or 95% or 96% or 97% or98% or 99% identical to a target site identified in the instantdisclosure. For example, the target sequences identified as SEQ ID)NO:AH and SEQ ID NO:AM are homologous sequences in related HPV strains(HPV16 and HPV11, respectively). Consequently, target sites in relatedstrains of HPV or HBV are identifiable based on their homology.Homologous sites may also be identified by aligning the proteinsequences of related viruses and then aligning the underlying nucleicacid sequences to find homologous or corresponding target sites.

[0125] 5.1.7. Ribozymes

[0126] The present invention provides methods by which a trans-actingribozyme may be used in addition to the toxic agents of the invention.Further, a multi-ribozyme may be used as an expression system for one ormore toxic agents or trans-acting ribozymes. These ribozymes of theinvention can be used, for example, to destroy tissue-specific disease,or to treat bacterial, viral, or parasitic infections. The ribozymes ofthe present invention may comprise one or more multi-ribozymes.

[0127] In accordance with the present invention, the multi-ribozyme maycomprise one or more ribozymes or one or more ribozyme cassettes. Eachcassette in turn may consist of a catalytic core (e.g., containing oneor more trans-acting ribozymes or containing one or more toxic agents)and one or more flanking regions. The catalytic core can target apathogen, by specifically inhibiting a pathogen-specific target. Thecatalytic core can target a cell (such as a diseased cell), byspecifically inhibiting a tissue-specific target (such asdisease-specific target). Further, as described in sections below, themulti-ribozymes of the invention also provide a means of deliveringtoxic agents to a cell, and expressing toxic agents of the invention(including antisense RNA, toxic gene products) in a cell ortissue-specific, or pathogen-specific manner. In one embodiment, theribozyme cassette may consist of a 5′ autocatalytically cleavingribozyme sequence, a core catalytic ribozyme comprising a trans-actingribozyme and a 3′ autocatalytically cleaving ribozyme. In anotherembodiment, the multi-ribozymes comprise a cassette including, theenhanced 5′ and 3′ autocatalytically cleaving ribozyme sequence. Inanother embodiment, the multi-ribozymes contain one or more internaltrans-acting ribozymes. Such trans-acting ribozymes may be directed tothe same site on the same RNA, different sites on the same RNA, ordifferent RNAs. Thus, trans-acting ribozymes of the invention may targeta pathogen-specific RNA or tissue-specific RNA.

[0128] The present invention also provides multi-ribozymes and their useto target RNA in a tissue-specific or pathogen-specific manner for thetreatment of disease such as bacterial infection. The invention providesmulti-ribozymes containing one or more internal trans-acting ribozyme.Trans-acting ribozymes act in a target-specific manner and thereforemay, in certain embodiments, act on a pathogen (such as bacteria) or aselected cell (such as a diseased cell) to enhance the use of toxicagent. In accordance with the present invention, the multi-ribozymes maycomprise a) a trans-acting ribozyme or toxic agent flanked by 5′ and 3′autocatalytically cleaving ribozymes or enhanced autocatalyticallycleaving ribozymes; b) a trans-acting ribozyme or toxic agent flanked byeither a 5′ or 3′ autocatalytically cleaving ribozyme; or c)multi-transacting ribozymes and/or multiple toxic agents, flanked by oneor both 5′ and 3′ autocatalytically cleaving ribozymes or enhancedautocatalytically cleaving ribozymes. For example, in a specificembodiment, the invention provides a multi-ribozyme with twotrans-acting ribozymes, wherein the first trans-acting ribozyme cleavesan HBV target, and the second trans-acting ribozyme cleaves a HCVtarget. In this embodiment, it may also be desirable to target theexpression of such multi-ribozyme to the liver, e.g., by operativeassociation with a liver-specific promoter. Thus, the multi-ribozymes ofthe invention may be used to deliver one or more toxic agents to abacteria or bacteria-infected cell or tissue. In accordance with thepresent invention the multi-transacting ribozymes may be targeted to thesame site on the same RNA, different sites on the same RNA or differentRNAs. In accordance with the present invention the multiple toxic agentsmay be targeted to the same site on the same target (such as a cellularRNA or protein), different sites on the same target or differenttargets.

[0129] The ribozymes of the present invention possesses sufficientcatalytic activity to inactivate the RNA of the targeted RNAs. From anantimicrobial perspective, hammerhead-type ribozymes are especiallyattractive since the molecule inactivates gene expression catalyticallythrough the cleavage of the phosphodiester bond of the mRNA.Furthermore, hammerhead-type ribozymes have been re-engineered tofunction in an intermolecular or transducer (trans) acting state(Haseloff et al., 1988, Nature 334(6183):585-91; Uhlenbeck. O. C., 1987,Nature 328(6131):59). The catalytic activity of the ribozyme requires asufficient concentration of the divalent cation, Mg⁺², and substrate.The substrate can have any sequence as long as the cleavages sitecontains the recognition element NUX, where N represents any nucleotide,U corresponds to uracil, and X is any nucleotide except G (Koizumi etal., 1989, Nucleic Acids Resonant. 17(17):7059-71). Ribozymes have beenwidely demonstrated to function in vivo (Christoffersen et al., 1995, J.Med. Chem. 38(12):2023-37; Inokuchi et al., 1994, J. Biol. Chem.269(15):11361-6). The present invention improves the initial design ofhammerhead-type ribozymes (Taira et al., 1991, NAR 19(9):5125-5130) byconstructing multi-ribozymes consisting of ribozyme cassettes. Ribozymecassettes contain one or more cis-acting hammerhead ribozymes flanking aribozyme that inactivates the targeted RNA(s) as well as one or moreflanking sequences. Upon transcription the targeted ribozyme is releasedas a 60-70 base transcript which not only improves its specificity byreducing non-specific interactions but also improves its catalyticactivity as well. This invention includes modifications to and use ofthe ribozyme described in U.S. Pat. No. 5,824,519 and PCT publicationsNo. WO98/24925, WO97/17433, WO98/24925, WO99/67400, which areincorporated by reference herein in their entirety.

[0130] 5.2. Nucleic Acids Encoding Toxic Agents or Ribozymes

[0131] The invention also provides nucleic acids and expressioncassettes which encode the ribozymes and/or toxic agents of theinvention. These nucleic acids can be used to express the ribozymes ortoxic agents of the invention at the selected site. The site can betissue-specific in the case of treating tissue-specific cancers ordisease, or it can be pathogen-specific in the case of ribozymes ortoxic agents that prevent replication of infectious agents to treatinfection (e.g., hepatitis, herpes, malaria, tuberculosis, bacterialinfections etc.). The invention provides nucleic acids which encodetoxic agent(s) and/or ribozyme(s) which are target-specific. Theinvention also provides nucleic acids which encode toxic agent(s) and/orribozyme(s) operably linked to a tissue-specific or pathogen-specificpromoter.

[0132] There are several options for constructing the multi-ribozymeencoding sequences: 1) ribozymes directed to different targets in thesame pathogen 2) multiple copies of the same ribozyme 3) multipleribozymes directed to multiple targets, and 4) multiple ribozymesdirected to different sites on the same target. There are also severaloptions for constructing the toxic agent encoding sequences: 1) toxicagents directed to different targets in the same pathogen 2) multiplecopies of the same toxic agent 3) multiple toxic agents directed tomultiple targets, and multiple toxic agents directed to the same target.Further, toxic agents and ribozymes may be combined in various ways,e.g., a multi-ribozyme and a nucleic acid encoding a toxic agent may beengineered in a single construct under one promoter. The promoter canhave the chosen level of specificity as described herein.

[0133] The nucleic acids of the invention encode one or more toxicagents of the invention. Thus, nucleic acids encoding toxic proteins ofthe invention include but are not limited to addiction system toxins.The invention further provides modified and enhanced addiction systemtoxins which have been engineered to be more toxic or more specific to aparticular target pathogen. The present invention provides nucleic acidsencoding antisense molecules targeted to RNA of a gene which plays acritical role in the survival of the pathogen, or which is essential tothe pathogen's life cycle. The present invention also encompassesnucleic acids comprising modifications to naturally occurring antisensemolecules which modulate the expression of an essential gene product ofa pathogen.

[0134] The nucleic acids of the invention also relate to those encodingantisense molecules of the invention. The invention provides modifiedand enhanced antisense molecules which have enhanced stability, enhancedcomplementarity to a target RNA, or enhanced specificity for a targetRNA or target pathogen. The invention also provides nucleic acidsencoding modified naturally occurring antisense molecules, such asnucleotide sequences which have similar functions as DicF in modulatingthe expression of gene products essential to the pathogen's life cycleor survival.

[0135] The nucleic acids of the invention also relate to nucleic acidsencoding sense RNA molecules capable of targeting an essential antisensemolecule.

[0136] The nucleic acid, encoding a toxic agent selected from the groupconsisting of ccdB, kid, perK, parE, doc, higB, clipAK, chpBK, kicB,hoc, srnB′, flmA, pmdA, relF, gef kilA, kilB, kilC, kilE, traL, traE,sigB, hok, pemK, lysostaphin, and kikA is provided. The nucleic acidencoding the toxic agent DicF1, or DicF1-like, is provided.

[0137] In several embodiments, nucleic acids of the invention encode acatalytic multi-ribozyme(s) that contains two separable functionalregions including a) a catalytic sequence (also known as the “catalyticcore”) which cleaves the target RNA, and b) flanking regions whichinclude cis-acting autocatalytically cleaving ribozyme(s). As describedabove, the catalytic core consists of one or more trans-actingribozyme(s) and/or one or more toxic agent(s). The present inventionprovides nucleic acid which encode an internal targeted ribozymecontaining two or more trans-acting ribozymes, wherein each of theseparate trans-acting ribozymes can be targeted to the same or differenttarget RNA molecules. By nucleic acid complementarity, the binding sitedirects the ribozyme core to cleave a specific site on the target RNAmolecule. The length of flanking sequences have implications not onlyfor specificity, but also for the cleavage efficiency of the individualribozyme molecules. In the present catalytic ribozyme, the flankingsequences are highly specific for the target RNA, yet allow readydissociation from the target RNA once cleavage occurs. This permitscycling of the ribozyme and reduces the amount of ribozyme required tobe effective. A range of binding/dissociation values from 16-21 Kcal isexpected to be effective. The present invention provides nucleic acidwhich encode a two or more toxic agents, wherein each of the toxicagents can be targeted to the same or different target molecules.

[0138] The invention additionally provides nucleic acids and expressioncassettes which encode the toxic agent and/or ribozymes of theinvention. These nucleic acids can be used to express the toxic agentand/or ribozyme of the invention at the selected site. In oneembodiment, the nucleic acid comprise a tissue-specific promoteroperably linked to a toxic agent. In another embodiment, the nucleicacids and expression cassettes of the invention comprise atissue-specific promoter operably linked to a sequence encoding acatalytic ribozyme comprising one or more target RNA-specifictrans-acting ribozymes and one or more toxic agents. In anotherembodiment, the nucleic acids comprise a pathogen-specific promoter froma sequence encoding a toxic agent. In another embodiment, the nucleicacids and expression cassettes of the invention comprise apathogen-specific promoter operably linked to a sequence encoding a 5′autocatalytically cleaving ribozyme sequence, a catalytic ribozymecomprising one or more target RNA-specific trans-acting ribozymes and/orpathogen-specific toxic agents, and a 3′ autocatalytically cleavingribozyme sequence. In accordance with the present invention, theexpression cassettes may be engineered to express two or moremulti-ribozymes containing trans-acting ribozymes which act on the sameor different targets. The expression cassettes may also be engineered toexpress two or more multi-ribozymes containing 5′ and 3′autocatalytically cleaving ribozymes with either slow or enhancedcleavage activity.

[0139] The expression cassettes of the invention or the nucleic acidsencoding the toxic agents of the invention may be placed into anysuitable plasmid known in the art (such as a bacteriophage transferplasmid, bacterial plasmid, or eukaryotic expression plasmid). Theinvention also provides novel and modified plasmids for use inaccordance with the invention.

[0140] At the molecular genetic level the coding sequence for a toxicagent, ribozyme, or multi-ribozyme of the invention may be placed underthe control of one or more of the following genetic elements: anaturally occurring strong, intermediate, or weak constitutivelyexpressed or regulated promoter from the targeted microorganism, or anartificially contrived constitutively expressed or regulated promotercontaining either a strong, intermediate or weak consensus sequence thataccords the desired levels of ribozyme and/or toxic agent expression.The present invention provides promoter elements which arepathogen-specific. The invention provides promoter elements which areused to achieve pathogen-specific expression of the toxic agents of thepresent invention. The present invention provides promoter elementswhich are tissue-specific. The invention provides promoter elementswhich are used to achieve tissue-specific expression of the toxic agentsof the present invention. Accordingly, the present invention providesnucleic acids encoding promoter elements which are pathogen-specific.The invention provides promoter elements which are used to achievepathogen-specific expression of the toxic agent(s) and/or ribozyme(s) ofthe present invention. The present invention provides promoter elementswhich are tissue-specific. The invention provides promoter elementswhich are used to achieve tissue-specific expression of the toxicagent(s) and/or ribozyme(s) of the present invention.

[0141] In one embodiment, the nucleic acid comprise a tissue-specificpromoter operably linked to a sequence encoding one or more toxicagent(s). In another embodiment, the nucleic acids comprise atissue-specific or pathogen-specific promoter operably linked to asequence encoding at least one autocatalytic ribozyme and one or moretrans-acting ribozymes. In another embodiment, the nucleic acidscomprise a tissue-specific or pathogen-specific promoter operably linkedto a sequence encoding at least one or more toxic agents. In anotherembodiment, the nucleic acids comprise a pathogen-specific promoteroperably linked to a sequence encoding at least one autocatalyticribozyme and one or more trans-acting ribozymes and one or more toxicagents. In accordance with the present invention, the trans-actingribozymes and/or toxic agents of the invention may act on the same ordifferent targets.

[0142] In yet another embodiment, the present invention provides a novelvector or plasmids encoding the toxic agent(s) and/or ribozyme(s) of theinvention. The novel vectors of the present invention may be used toengineer a wide variety of toxic agents and/or ribozymes including, butnot limited to, tissue-specific, pathogen-specific, promoter-specific,antimicrobial specific, antiviral specific, anticancer specific,antitumor specific, or target-specific. The invention also relates to avector or plasmid origin of replication which modulates specificity ofthe replication of a vector or plasmid in a cell or pathogen. Theinvention also relates to the copy number of a vector or plasmid in aselected cell or pathogen to modulate the dose of the toxic agent and/orribozyme.

[0143] In a specific embodiment, the invention provides novel plasmidswhich encode a toxic protein. In another specific embodiment, theinvention provides novel plasmids which encode a mutant bacteriophagepac site or a mutant bacteriophage pacABC sequence.

[0144] 5.2.1. Eucaryotic and Procaryotic Expression Vectors

[0145] The present invention encompasses expression systems, botheucaryotic and procaryotic expression vectors, which may be used toexpress the toxic agents and/or multi-ribozymes of the invention. TheDNA expression vectors and viral vectors containing the nucleic acidsencoding the toxic agents of the present invention may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing the expression vectors and viral vectors of theinvention by expressing nucleic acid encoding a toxic agent and/ormulti-ribozyme sequences are described herein. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing gene product coding sequences and appropriatetranscriptional and translational control signals. These methodsinclude, for example, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, supra, and Ausubel etal., 1989, supra. Alternatively, nucleic acids capable of encoding atoxic agent and/or ribozyme sequence may be chemically synthesizedusing, for example, synthesizers. See, for example, the techniquesdescribed in “Oligonucleotide Synthesis”, 1984, Gait, M. J. ed., IRLPress, Oxford, which is incorporated by reference herein in itsentirety.

[0146] A variety of host-expression vector systems may be utilized toexpress the selected toxic agent and/or multi-ribozyme of the invention.Such host-expression systems represent vehicles by which the sequencesencoding the toxic agents or ribozymes of the invention may beintroduced into cells, tissues, or pathogens both in vivo and in vitrobut also represent cells which may, when transformed or transfected withthe appropriate nucleotide coding sequences, to express a toxic agentand/or ribozymes of the invention. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining selected toxic agent(s) and/or multi-ribozyme codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing the selected toxicagent(s) and/or multi-ribozyme coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the selected toxic agent(s) and/or multi-ribozyme codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing selected toxic agent(s) and/or multi-ribozymecoding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293,3T3) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter).

[0147] 5.3. Delivery and Expression of Toxic Agents

[0148] The invention also provides a novel vehicle for the delivery oftoxic agents or ribozymes of the invention. The invention encompassesDNA expression vectors and viral vectors that contain any of theforegoing coding sequences operatively associated with a regulatoryelement that directs expression of the coding sequences and geneticallyengineered host cells that contain any of the foregoing coding sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences or RNAs in the host cell or pathogen.A key to the present invention is the strategy used to deliver the toxicagent and/or ribozyme to the targeted microorganism or pathogen. Twoseparate classes of delivery systems can be manufactured, one biologicin nature and the other abiologic.

[0149] Accordingly, present invention also provides the delivery of thetoxic agents of the invention to cell or pathogen by abiologic orbiologic systems. The present invention provides compositions of matterwhich has resulted from the development of methods and compositions forthe delivery of one or more ribozymes and/or toxic agents directedagainst fundamental and essential cellular processes specific to atargeted microorganism through an inactivated, altered, or modifiedvirus (virion) or bacteriophage delivery vehicles. The present inventionalso provides abiologic delivery vehicles, capable of delivering anucleic acid comprising the toxic agent(s) and/or ribozyme(s) into thetargeted microorganism.

[0150] 5.3.1. Biologic Delivery Vehicles

[0151] The biologic delivery vehicle of the invention takes advantage ofthe fact that generalized transducing particles lack DNA originatingfrom the viral delivery vehicle or have a reduced capacity to transferDNA originating from the viral delivery vehicle. In a preferredembodiment of the invention, the viral delivery vehicle is abacteriophage, or modified bacteriophage. In one embodiment, such viralor bacteriophage particles only contain sequences of host origin. Inother embodiments, such particles contain engineered plasmids/vectorsencoding the toxic agent(s) or ribozyme(s) to be delivered. In otherembodiments, such particles contain engineered plasmids/vectors encodingthe toxic agent(s) or ribozyme(s) to be delivered and contain mutationswhich inactivate the ability of the delivery vehicle to transfer DNAoriginating from the delivery vehicle. Consequently, the invention usesa biologic assembly of viral head proteins (packaging elements for theantimicrobial therapeutic) around the nucleic acid containing thenecessary genetic elements that will insure the desired level ofexpression of the toxic agent(s) and/or ribozyme(s). An importantfeatures of the present invention are the combination of toxic agents orribozyme with viral delivery and assembly of the virions using a uniquecombination of plasmid features.

[0152] In one preferred embodiment, the invention provides bacteriophagewhich deliver a toxic agent of the invention. Bacteriophage of theinvention may be constructed to deliver one or more toxic agents of theinvention, such as one or more toxic gene products, proteins, antisenseRNAs, sense RNAs, or combination thereof. In another embodiment of theinvention, a host cell is constructed to express a pathogen-specifictoxic agent or ribozyme. In yet another embodiment of the invention, ahost cell is constructed to express a repressor of a promoter used inthe invention.

[0153] In other embodiments, a host cell may be engineered tooverexpress an antidote to a toxic agent such that the host cell isprotected from toxicity and may be used as a producing strain, ormanufacturing strain.

[0154] The present invention also encompasses expression systems, whichmay be used to express the toxic agents and/or ribozymes such asbacteriophage, viral vectors, etc. For example, a variety ofbacteriophage systems may be utilized to express the selectedribozyme(s) and/or toxic agent(s) of the invention. For example, suchbacteriophage systems represent vehicles by which the sequences encodingthe toxic agent(s) and/or ribozyme(s) may be introduced into targetbacteria both in vivo and in vitro. In several embodiments, the specificbacteriophage which is selected determines the species of bacteria whichis targeted and infected by that bacteriophage.

[0155] In one embodiment, delivery of a toxic agent to a pathogen is byuse of a bacteriophage or other delivery vehicle which targets thepathogen of interest. In one embodiment, the bacteriophage (or deliveryvehicle) delivers the toxic agent or nucleic acids encoding the toxicagent to the pathogen. In a specific embodiment, a toxic agent of theinvention is delivered to a bacterial cell by a modified bacteriophagecapable of infecting a pathogenic bacteria. In a further embodiment,bacteriophage are selected for their ability to infect a particularspecies or genera of bacteria, and are used to deliver a toxic agent forthe eradication of such bacterial species or genera from a host. In apreferred embodiment, the delivery vehicle or nucleic acids native tothe delivery vehicle are modified such that they contain insufficientgenetic information for the delivery of nucleic acids native to thedelivery vehicle. Thus, the modified delivery vehicle (e.g. virion orbacteriophage) can serve as a molecular vehicle that delivers theribozyme(s) and/or toxic agent(s) of the invention to the target cell orpathogen, but does not deliver replicable nucleic acids native to thedelivery vehicle. Alternatively, an abiologic delivery system e.g.,liposomes) can be used to package nucleic acid carrying the geneticelements necessary and sufficient for the proper expression of theribozyme(s) and/or toxic agent(s).

[0156] The toxic agents and/or ribozymes of the invention may be used totreat infection from a variety of pathogens. These include but are notlimited to microorganisms such as bacteria, parasites, and fungi. Inspecific embodiments of the invention, the toxic agents of theinvention, delivered by a viral delivery vehicle (such as a modifiedbacteriophage are useful to treat microbial infections associated withsevere burns, cystic fibrosis, cancer, or other immunocompromisingconditions.

[0157] 5.3.1.1. Delivery & Expression by Viral Vectors

[0158] In accordance with the present invention, a wide variety ofviruses and viral vectors may be used to deliver the nucleotidesequences encoding the toxic agent(s) and/or ribozymes of the presentinvention, a few examples of which are described below. In this regard,a variety of viruses may be genetically engineered to express theselected toxic agent(s) and/or ribozymes in order to target a specificpathogen.

[0159] The present invention also relates to the delivery of the toxicagents of the invention to cell or pathogen by abiologic or biologicsystems. In a specific embodiment, For example, as described herein, atoxic agent of the invention is delivered to a bacterial cell by abacteriophage capable of infecting a pathogenic bacteria. In a furtherembodiment, bacteriophage are selected for their ability to infect aparticular species of bacteria, and are used to deliver a toxic agentfor the eradication of such bacterial species from a host.

[0160] The invention provides for use of a virion which can also be anybacteriophage which specifically infects a bacterial pathogen of thepresent invention as well as any virus which can be specificallytargeted to infect the pathogen of the present invention. For example,the bacteriophage can include, but is not limited to, those specific forbacterial cells of the following genera: Bacillus, Campylobacter,Corynebacterium, Enterobacter, Enterococcus, Escherichia, Klebsiella,Mycobacterium, Pseudomonas, Salmonella, Shigella, Staphylococcus,Streptococcus, Vibrio, Streptomyces, Yersinia and the like (see, e.g.,the American Type Culture Collection Catalogue of Bacteria andBacteriophages, latest edition, Rockville, Md.), as well as any otherbacteriophages now known or later identified to specifically infect abacterial pathogen of this invention. The invention also provides forthe use of a virion which specifically infects a fungal pathogen.

[0161] This delivery system consists of a DNA plasmid carrying thenucleic acids coding for the toxic agent(s) and/or ribozyme(s) packagedinto viral particles. Specificity is conferred by the promoter drivingtranscription of the toxic agents and/or ribozymes and by the hostspecificity of the viral vehicle. Specificity is also conferred by theorigin of replication controlling vector replication.

[0162] In the virions of the present invention, the non-viral DNA canencode one or more toxic agent(s) and/or one or more ribozyme(s). In thevirions, the non-viral DNA can comprises a pathogen-specific ortissue-specific promoter operably linked to a sequence encoding one ormore toxic agents or ribozymes.

[0163] The nucleic acid delivered by a virion can encode one or moretoxic agent(s) and/or one or more ribozyme(s) or a combination thereof.The virion can comprise any nucleic acid encoding a ribozyme or toxicagent, particularly those described herein.

[0164] Bacteriophage P1

[0165] The invention provides the use of any virion for the delivery ofa toxic agent or ribozyme to a target cell. For example, a commonbacteriophage of E. coli, P1, is an attractive delivery vehicle for theinvention for a number of reasons. First and foremost, P1 has a broadintergenera and interspecies range (Yarmolinsky et al., 1988, Mol. Gen.Genet. 113:273-284). The P1 receptor of E. coli is the terminal glucoseof the lipopolysaccharide (LPS) core lysergic ring of the bacterialouter membrane (Generalized Transduction, p. 2421-2441. In F. Neidhardt(ed.), Escherichia coli and Salmonella:Cellular and Molecular Biology,2d ed. Vol.2, ASM Press, Washington, D.C.). Yarmolinsky and Sternbergreport that in addition to E. coli, this particular phage has theability to inject its nucleic acid into a large number (>25) of diverseGram negative bacteria (Yarmolinsky et al., 1988, Mol. Gen. Genet.113:273-284). Secondly, P1 can accommodate a significant amount ofgenetic information, over 2% (100,000 bp) of the DNA of E. coli(Generalized Transduction, p. 2421-2441. In F. Neidhardt (ed.),Escherichia coli and Salmonella:Cellular and Molecular Biology, 2d ed.Vol.2, ASM Press, Washington, D.C.). Consequently, gene dosage of theribozymes or toxic agents can be increased through multiplication of thetoxic agents and/or ribozymes, thereby increasing the microbicidalactivity of the toxic agents and/or ribozymes. Accordingly,bacteriophage P1 is used as the delivery vehicle or molecular syringe.P1 has advantages over certain with lytic phage therapy which may harborrisk of 1) dissemination of undesirable products (e.g., DNA originatingfrom the P1 bacteriophage) to nonpathogenic indigenous microflora, 2)excessively narrow host range, 3) rapid clearance of the material by thereticuloendothelial system of the host and 4) the concern that a lyticinfection could become uncontrolled in commensal bacteria inimmunocompromised patients. In certain of these embodiments, the P1delivery system is the preferable delivery vehicle for delivery of atoxic agent to a target pathogenic bacterium. An additional advantage ofthe P1 delivery vehicle is that phage-mediated transfer of undesirableproducts may be decreased or avoided when the phage are engineered suchthat they are incapable of transferring endogenous phage DNA to thehost. In this embodiment, the phage particles inject transfer plasmidDNA into target bacterial cells. Expression of the encoded toxic agentsmay then result in bacterial cell death independent of the bacterium'sresistance to antibiotics.

[0166] Additionally, a process utilizing in vitro packaging is alsopossible. In vitro packaging can be accomplished through the addition ofPAC-sites to the genetic information of the toxic agent or ribozymeconstruct. P1 packaging initiates within one of the P1 PAC genes(Steinberg, N., 1987, J. Mol. Biol. 194(3) :469-79). It has beenreported that the active PAC site is contained within a 161 base-pairsegment of the P1 EcoR1 fragment 20 (Steinberg, N.,1987, J. Mol. Biol.194(3) :469-79). Thus, the phage head serves as a molecular syringe thatdelivers the inactivating ribozyme(s) and/or toxic agent(s) to thepathogen.

[0167] In specific preferred embodiments of the invention, a toxic agentis encoded in a Transfer plasmid, and is used in connection with a P1bacteriophage delivery system. Such Transfer plasmid preferablycontains 1) an origin or replication 2) selectable marker 3) Pac ABCgenes with a P1 PAC site 4) P1 lytic replicon and 5) nucleic acidsencoding one or more toxic agents of the invention (e.g., antisensemolecule, ribozyme, or toxic protein, etc). The Transfer plasmid may beproduced in a bacterium producing cell (e.g., a P1 lysogen). Inpreferred embodiments of the invention, the bacteriophage P1 plasmid(e.g., the P1 prophage) is engineered to be incapable of being packagedinto a phage head. In this embodiment, only Transfer plasmids arepackaged into virions. Such inhibition of P1 plasmid packaging isaccomplished by introducing a mutation or deletion in the P1 plasmidthat inhibits the P1 plasmid from being packaged into a virion or phagehead. Mutation(s) or deletion(s) of the P1 plasmid which inhibitpackaging include but are not limited to one or more mutations and/ordeletions in the P1 plasmid PAC site. Any mutation(s) and/or deletion(s)of the P1 plasmid which inhibits packaging of the bacteriophage P1plasmid is with in the scope of the invention. Such mutations ordeletions are introduced by standard techniques known in the art. Inseveral embodiments, the P1 lysogen has a temperature sensitiverepressor mutation (e.g. C1.100). Preferably, induction of the P1lysogen leads to the production of P1 phage heads containing only thepackaged Transfer plasmid. Bacteriophage containing the packagedTransfer plasmid nucleic acids may then be used to infect a target cellsuch as a bacterial pathogen. The bacteriophage infects a bacterialpathogen by injecting its nucleic acids into the bacterium. The toxicagent encoded in the bacteriophage nucleic acids is thus delivered tothe bacterium. Within the bacterium, the Transfer plasmid nucleic acidsrecircularize, and the toxic agent is expressed in the bacterium leadingto toxicity and death. Similar mutation and/or deletion strategies maybe used with the other viral delivery systems of the invention such thatthe deletion(s) and/or mutation(s) allow packaging of the nucleic acidsencoding toxic agent or ribozyme of the invention, but prevent packagingof nucleic acids encoding one or more viral genes or plasmids. Suchstrategies allow for construction of viral delivery systems which haveincreased safety (e.g., when used in connection with therapeutics of theinvention).

[0168] In a specific embodiment, the invention provides a bacteriophageable to package/deliver Transfer plasmid in P1 virions which will infecta pathogenic bacterial target. In another specific embodiment,bacteriophage P1 (PAC site) knockouts able to package/deliver Transferplasmid DNA but unable to incorporate P1 DNA thus preventing horizontaltransfer of undesirable products to non-pathogenic indigenousmicroflora. In another specific embodiment of the invention, the phagedelivery system comprises a Transfer plasmid carrying the genes encodingthe antimicrobial agents, a plasmid origin of replication, the P1 lyticorigin of replication and a minimal PAC site (e.g., such as the minimalP1 pac site as shown in FIG. 12, SEQ ID NO:7). In this embodiment, theplasmid is maintained in a bacteriophage P1 lysogen unable to packageits own DNA. The defective lysogen provides all the replication factorsneeded to activate the P1 origin of replication on the transfer plasmidand all the structural components necessary to form mature virions. Thelysogen also carries the c1.100 temperature-sensitive repressormutation. C1 is responsible for the repression of functions leading tovegetative phage production. Induction of the lysogen by a temperatureshift results in multiplication of DNA, packaging of the transferplasmid into P1 phage heads and lysis of the production strain. Virionsare harvested and used to deliver the Transfer plasmid to the pathogen.The phagehead contains multiple copies of Transfer plasmid DNA and istargeted to pathogenic bacteria by the bacteriophage's natural receptormediated mechanisms. Upon delivery, plasmid DNA recircularizes andexpression of the toxic agent under the control of environmental,virulence-regulated or species-specific promoters results in rapid celldeath.

[0169] In specific embodiments, the invention provides novel Transferplasmids encoding toxic agents which may be used in combination with abacteriophage delivery system in order to treat a bacterial infection ina host.

[0170] Bacteriophage Lambda

[0171] Another example of a system using bacteriophage virions topackage DNA carrying ribozymes and/or toxic agents directed against E.coli is the bacteriophage lambda. Similar strategies are used togenerate virions capable of delivering ribozymes and/or toxic agentsdirected against other microorganisms. The virions used to package theDNA can be species-specific, such as the virion derived from thebacteriophage lambda coat, or they can possess a broader host range,such as virion derived from bacteriophage P1, as described above. Broadhost-range viruses facilitate production of the antimicrobial agentswithout the loss of species specificity because species-specificpromoters are used to direct the transcription of the ribozymes whichare directed against species-specific targeted RNA sequences. Forexample, a lambda bacteriophage entails the use of a plasmid carryingthe ribozyme and/or toxic agent, a plasmid origin of replication, aselectable marker for plasmid maintenance, the minimal lambda origin ofreplication, and cos sites, which are required for packaging of DNA intolambda virions. This plasmid is maintained in a lambda lysogen that isdefective in integration/excision and recombination functions. Thedefective lysogen provides all of the replication factors needed toactivate the lambda origin of replication on the plasmid and all of thestructural components needed to form mature virions; however, thelysogen is not able to replicate and package its own DNA into thevirions. The lysogen may also carry a temperature-sensitive repressormutation (such as the cI857).

[0172] Other Viral Vectors

[0173] Retroviral vectors are also commonly used to deliver genes tohost cells both in vivo and ex vivo. Retroviral vectors are extremelyefficient gene delivery vehicles that cause no detectable harm as theyenter the cells. The retroviral nucleic acid may integrate into hostchromosomal DNA allowing for long-term persistence and stabletransmission to future progeny, such a vector would be useful for thedelivery of a toxic agent and/or ribozyme(s) used to target a cellulargene product involved in a chronic or hereditary disorder or to target aviral gene or a microbial gene or a parasitic gene involved in a chronicor persistent infection. An example of an appropriate retroviral vectorare, lentiviruses which have the advantage of infecting and transducingnon-dividing cells. In such an embodiment, a lentiviral vector encodinga packagable RNA vector genome operably linked to a promoter in whichall the functional retroviral auxiliary genes are absent, is used totransfer the DNA encoding the toxic agent and/or ribozyme of the presentinvention. Examples of such vectors are described in WO 98/17815, WO98/17816 and WO 98/17817, each of which is incorporated herein byreference in their entirety.

[0174] In yet another embodiment, non-integrating viral vectors whichinfect and transduce non-dividing cells, such as adenoviral vectors maybe used to deliver the toxic agent and/or ribozymes of the presentinvention. Adenoviral vectors have several advantages because the use ofsuch vectors avoids risks associated with permanently altering the hostcell genome or of promoting insertional mutagenesis. Adenoviruses areone of the best developed non-integrating viral vectors and can be usedto transfer expression cassettes of up to 75 kb. Recombinantadenoviruses can be produced at very high titers, is highly infectiousand efficiently transfers genes to a wide variety of non-replicating andreplicating cells and is ideal for in vivo mammalian gene transfer.

[0175] Adenovirus-based vectors are relatively safe and can bemanipulated to encode the desired toxic agent and/or ribozymes and atthe same time to be inactivated in terms of their ability to replicatein a normal lytic viral life cycle. Adenovirus has a natural tropism forairway epithelia. Therefore, adenovirus-based vectors are particularlypreferred for respiratory gene therapy applications. In a particularembodiment, the adenovirus-based gene therapy vector comprises anadenovirus 2 serotype genome in which the E1a and the E1b regions of thegenome, which are involved in early stages of viral replication havebeen deleted and replaced by nucleotide sequences of interest. In afurther embodiment, the adenovirus-based gene therapy vector containsonly the essential open reading frame (ORF3 or ORF6 of adenoviral earlyregion 4 (E4) and is deleted of all other E4 open reading frames, or mayadditionally contain deletions in the E3 regions (e.g., see U.S. Pat.No. 5,670,488, incorporated herein by reference in its entirety). Inanother embodiment, the adenovirus-based therapy vector used may be apseudo-adenovirus (PAV), which contain no harmful viral genes and atheoretical capacity for foreign material of nearly 36 kb.

[0176] In another embodiment, adeno-associated virus (AAV) systems maybe used to deliver the toxic agent and/or ribozymes of the presentinvention. AAV has a wide host range and AAV vectors have currently havebeen designed which do not require helper virus. Examples of such AAVvectors are described in WO 97/17458, incorporated herein by referencein its entirety.

[0177] Vaccinia viral vectors may be used in accordance with the presentinvention, as large fragments of DNA are easily cloned into its genomeand recombinant attenuated vaccinia variants have been described (Meyer,et al., 1991, J. Gen. Virol. 72:1031-1038). Orthomyxoviruses, includinginfluenza; Paramyxoviruses, including respiratory syncytial virus andSendai virus; and Rhabdoviruses may be engineered to express mutationswhich result in attenuated phenotypes (see U.S. Pat. Ser. No. 5,578,473,issued Nov. 26, 1996). These viral genomes may also be engineered toexpress foreign nucleotide sequences, such as the selected toxic agentand/or ribozymes of the present invention (see U.S. Pat. Ser. No.5,166,057, issued Nov. 24, 1992, incorporated herein by reference in itsentirety). Reverse genetic techniques can be applied to manipulatenegative and positive strand RNA viral genomes to introduce mutationswhich result in attenuated phenotypes, as demonstrated in influenzavirus, Herpes Simplex virus, cytomegalovirus and Epstein-Barr virus,Sindbis virus and poliovirus (see Palese et al., 1996, Proc. Natl. Acad.Sci. USA 93:11354-11358). These techniques may also be utilized tointroduce foreign DNA, i.e., the selected toxic agent and/or ribozyme,to create recombinant viral vectors to be used in accordance with thepresent invention. In addition, attenuated adenoviruses and retrovirusesmay be engineered to express the toxic agent and/or ribozymes.Therefore, a wide variety of viruses may be engineered to design theribozymes delivery vehicles of the present invention.

[0178] The viral vectors of the present invention may be engineered toexpress the toxic agents and/or ribozymes in a tissue specific manner.For example, the promoter of the carcinoembryonic antigen (LEA) isexpressed in a proportion of breast, lung and colorectal cancers, butrarely in healthy tissues. In order to target a hepatoma, theα-fetoprotein (AFP) promoter whose activity is restricted to malignantcells. Proliferating cells can be targeted with a flt-1 promoter, whichhas been shown to allow preferential targeting of proliferatingendothelial cells. See Miller et al., 1997, Human Gene Therapy8:803-815, incorporated herein by reference in its entirety.

[0179] 5.3.2. Abiologic Delivery Vehicles

[0180] Abiologic delivery of one or more toxic agents and/or ribozymesis accomplished by a variety of methods, including packaging plasmid DNAcarrying the gene(s) that codes for the toxic agent(s) and/orribozyme(s) into liposomes or by complexing the plasmid DNA carrying thegene(s) that codes for the toxic agent(s) and/or ribozyme(s) with lipidsor liposomes to form DNA-lipid or DNA-liposome complexes. The liposomeis composed of cationic and neutral lipids commonly used to transfectcells in vitro. The cationic lipids complex with the plasmid DNA andform liposomes.

[0181] A liposome is provided, comprising a nucleic acid comprising apathogen-specific promoter operably linked to a sequence encoding atrans-acting ribozyme comprising a) a 5′ autocatalytically cleavingribozyme sequence, b) a catalytic ribozyme comprising a targetRNA-specific binding site and c) a 3′ autocatalytically cleavingribozyme sequence.

[0182] A liposome is provided, comprising a nucleic acid encoding apathogen-specific promoter operably linked to a sequence encoding one ormore toxic agents is provided.

[0183] The liposome of the invention, wherein the nucleic acid encodesmore than one trans-acting ribozyme and/or more than one toxic agent isprovided. The liposome can comprise any ribozyme-encoding nucleic acid,or any toxic agent encoding nucleic agent particularly those describedherein. Such nucleic acids may be operably linked to a tissue-specificor pathogen-specific promoter.

[0184] The liposomal delivery systems of the invention can be used todeliver a nucleic acid comprising a tissue-specific promoter operablylinked to a sequence encoding a multi-ribozyme comprising a) a 5′autocatalytically cleaving ribozyme sequence, b) a catalytic ribozymecomprising a target RNA-specific binding site and c) a 3′autocatalytically cleaving ribozyme sequence.

[0185] The liposome delivery system of the invention can be used todeliver a nucleic acid comprising a tissue-specific promoter operablylinked to a sequence encoding one or more toxic agents. The liposomedelivery system of the invention can be used to deliver a nucleic acidcomprising a pathogen-specific promoter operably linked to a sequenceencoding one or more toxic agents.

[0186] Cationic and neutral liposomes are contemplated by thisinvention. Cationic liposomes can be complexed with a negatively-chargedbiologically active molecule (e.g., DNA) by mixing these components andallowing them to charge-associate. Cationic liposomes are particularlyuseful when the biologically active molecule is a nucleic acid becauseof the nucleic acids negative charge. Examples of cationic liposomesinclude lipofectin, lipofectamine, lipofectace and DOTAP (Hawley-Nelsonet al.,1992, Focus 15(3):73-83; Felgner et al., 1987, Proc. Natl. Acad.Sci. U.S.A. 84:7413; Stewart et al., 1992, Human Gene Therapy3:267-275). Procedures for forming cationic liposomes encasingsubstances are standard in the art (Nicolau et al., 1987, MethodsEnzymol. 149:157) and can readily be utilized herein by one of ordinaryskill in the art to encase the complex of this invention.

[0187] In yet another embodiment of the present invention, the plasmidDNA carrying the gene(s) that codes for the toxic agents and/orribozymes of the invention are complexed with liposomes using animproved method to achieve increased systemic delivery and geneexpression (Templeton et al., 1997, Nature Biotechnology 15: 647-652,incorporated herein by reference in its entirety). In accordance withthe present invention, an improved formulation of cationic lipids whichgreatly increase the efficiency of DNA delivery to host cells, withextended half-life in vivo and procedures to target specific tissues invivo. For example, but not by limitation, peptides and proteins may beengineered to the outer lipid bilayer, such as liver-specific proteins,leads to substantially enhanced delivery to the liver etc.

[0188] In one embodiment of the present invention, systemic delivery andin vivo and ex vivo gene expression is optimized using commerciallyavailable cationic lipids, e.g., dimethyldioctadeclammonium bromide(DDAB); a biodegradable lipid 1, 2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP); these liposomes may be mixed with a neutral lipid,e.g., L-αdioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol),two commonly used neutral lipids for systemic delivery. DNA:liposomeratios may be optimized using the methods used by those of skill in theart (e.g., see Templeton et al., 1997, Nature Biotechnology 15: 647-152,incorporated herein by reference in its entirety).

[0189] In yet another embodiment of the present invention, the plasmidDNA carrying the nucleic acids encoding the toxic agents and/orribozymes of the invention may be delivered via polycations, moleculeswhich carry multiple positive charges and are used to achieve genetransfer in vivo and ex vivo. Polycations, such as polyethilenimine, maybe used to achieve successful gene transfer in vivo and ex vivo (e.g.,see Boletta et al., 1996, J. Am. Soc. Nephrol. 7: 1728, incorporatedherein by reference in this entirety.)

[0190] The liposomes may be incorporated into a topical ointment forapplication or delivered in other forms, such as a solution which can beinjected into an abscess or delivered systemically, or delivered by anaerosol.

[0191] Plasmid DNA coding for the ribozymes or toxic agent is usedrather than preformed ribozymes or toxic agent for the followingreasons. Plasmid DNA allows the targeted cells to produce the toxicagent or ribozyme and, thus, results in a higher delivered dose to thecell than can be expected by delivery of ribozyme RNA or toxic agent vialiposome. The DNA also provides specificity of action based on targetsequence specificity. The liposomes deliver their DNA to any cell in thearea of administration, including cells of the host. The promoterdriving the transcription of the toxic agent or ribozyme is specific forthe targeted microorganism and, thus, will be inactive in other celltypes. Therefore, liposomal delivery of DNA coding for the toxic agentor ribozyme provides amplification and specificity. The presentinvention relates to promoter elements which are pathogen-specific ortissue-specific. Such promoter elements are used to achievepathogen-specific or tissue-specific expression of the toxic agent(s)and/or ribozyme(s) of the present invention. The invention also relatesuse of an origin of replication which modulates specificity of thereplication or copy number of a vector or plasmid in a cell or pathogen.

[0192]5.3.2.1. Delivery & Expression Using Multi-Ribozymes

[0193] In another embodiment of the invention, expression of a toxicagent is directed by a tissue-specific, pathogen-specific, and/ortarget-specific ribozyme or ribozyme cassette. The invention providesribozymes that have the unique characteristic of being both targetRNA-specific in their catalytic action, and tissue-specific orpathogen-specific in their expression. A ribozyme can be tissue-specificin the case of treating tissue-specific disease, or it can bepathogen-specific in the case of treating a pathogen such as E. coli.Multi-ribozymes may have one or more target-specific ribozyme(s) (e.g.,a trans-acting catalytic ribozyme) as well as elements which controltissue-specific or pathogen-specific expression.

[0194] In one embodiment, the nucleic acids of the invention comprise atissue-specific promoter operably linked to a sequence encoding a) a 5′autocatalytically cleaving ribozyme sequence, b) a catalytic ribozymecomprising a target RNA-specific binding site and c) a 3′autocatalytically cleaving ribozyme sequence.

[0195] The tissue-specific promoter in the ribozyme-producing constructresults in tissue-specific expression of the ribozyme in tissue(s) thatactively transcribe RNA from the selected promoter. Thus, only thetarget RNA in tissue that utilize the promoter will be cleaved by theribozyme.

[0196] Further, in accordance with the present invention, themulti-ribozyme may consist of one or more ribozyme cassettes. Eachcassette in turn may consist of a catalytic core and one or moreflanking sequences. In one embodiment, the ribozyme cassette may consistof a 5′ autocatalytically cleaving ribozyme sequence, a core catalyticribozyme comprising a trans-acting ribozyme and a 3′ autocatalyticallycleaving ribozyme. In yet another embodiment, the catalytic corecontains sequences encoding one or more toxic agent(s). In otherembodiments, the multi-ribozymes comprise a cassette including, theenhanced 5′ and 3′ autocatalytically cleaving ribozyme sequence. Inanother embodiment, the multi-ribozymes contain one or more internaltrans-acting ribozymes. In a preferred embodiment, the multi-ribozymesof the present invention include, but are no limited to triple ribozymecassettes. In another embodiment, multi-ribozymes include but are notlimited to one or more triple ribozyme cassettes linked together. In yetanother embodiment, the multi-ribozyme comprises a ribozyme cassettecontaining one or more internal trans-acting ribozyme. In an additionalembodiment, the multi-ribozyme comprises a series of one or moreribozyme cassettes containing one or more internal trans-actingribozymes or any combination thereof. In further embodiments, themulti-ribozyme cassettes or toxic agent(s) are expressed in atissue-specific or pathogen-specific manner. In a preferred embodimentof the invention, pathogen-specific expression is coupled to apathogen-specific promoter.

[0197] 5.4. Promoter Selection

[0198] Promoter selection is accomplished using techniques that areavailable in the art. As used herein, regulatory elements include butare not limited to, inducible and non-inducible promoters, enhancers,operators and other elements known to those skilled in the art thatdrive and regulate expression. Specifically, the invention providesinducible promoters which have increased transcriptional control andhigh expression levels. The promoter can be a naturally occurringstrong, intermediate or weak constitutively expressed or regulatedpromoter from the targeted microorganism, or an artificially contrivedconstitutively expressed or regulated promoter containing either astrong, intermediate or weak consensus sequence that delivers desiredlevels of toxic agent or ribozyme expression in the targeted microbe.

[0199] Promoters specific for the target (e.g., a specific pathogen,genus, etc.) in question can be selected by screening genomic sequencesfor the ability to activate a promoterless reporter gene. Thepromoterless reporter gene is based on the strategy developed for usewith plasmid pMC1871 (Casadaban et al., 1983, Meth. Enzymol. 100:293).For non-viral pathogens, plasmid capable of stable replication andmaintenance in the microorganism understudy is modified by standardmolecular biology techniques to carry the coding region of a reportergene (Sambrook et al. latest edition). The reporter gene can be any of anumber of standard reporter genes including but not limited to the lacZgene of E. coli, which codes for β-galactosidase. Total genomic DNA isisolated from cells of the pathogen, cleaved with restrictionendonucleases to yield fragments of a few hundred basepairs on average.These fragments are then ligated into a unique restriction endonucleasecleavage site at the 5′ end of the reporter gene coding region, creatinga library of plasmids. The library is then transformed into the pathogenby standard techniques and the resulting transformants are screened forexpression of the reporter gene. In the case of lacZ, the transformantscan be plated onto medium containing the chromogenic galactosidasesubstrate X-Gal (5-bromo-4-chloro-3-indolyl-D-galactoside).Transformants that contain a plasmid with an insert carrying a promoterwill express β-galactosidase and will turn blue on X-Gal plates. Theintensity of the blue color is relative to the level of expression;promoters of different strength can be selected based on the intensityof the blue color.

[0200] The above-described screening procedure can be modified toidentify regulated promoters. For example, promoters that are regulatedby carbon source availability can be screened on plates that containdifferent carbon sources. Other modifications are possible and willdepend, in part, on the organism in question. To test forspecies-specificity, the identified promoters are transferred topromoterless reporter plasmids capable of replication and maintenance ina different organism. Truly species-specific or pathogen-specificpromoters will not activate the expression of the reporter gene in anyother species. Obvious modifications can be used to identify and testartificial promoters composed of synthetic oligonucleotides insertedinto the promoterless reporter plasmid.

[0201] In one embodiment, the nucleic acids of the invention comprise atissue-specific promoter operably linked to a sequence encoding a 5′autocatalytically cleaving ribozyme sequence, one or more catalytictarget-specific trans-acting ribozymes or one or more toxic agents and a3′ autocatalytically cleaving ribozyme sequence.

[0202] The tissue-specific promoter in the ribozyme-producing constructresults in tissue-specific expression of the ribozyme in tissue(s) thatactively transcribe RNA from the selected promoter. Thus, only thetarget RNA in tissue that utilize the promoter will be cleaved by theribozyme. The pathogen-specific promoter binding site in theribozyme-producing construct results in pathogen-specific expression ofthe ribozyme in pathogens or microbes that actively transcribe RNA fromthe selected promoter. Thus, only the target RNA in pathogens thatutilize the promoter will be cleaved by the ribozyme.

[0203] Tissue-specific promoters can be used in the present nucleic acidconstructs. Examples of these promoters include the sequences forprobasin-promoter, a promoter-specific for prostate epitheliumprostate-specific antigen (prostate), keratin, k7 (epidermal sebaceousglands), albumin (liver), fatty acid binding protein (ileum), wheyacidic protein (breast), lactalbumin, smooth muscle actin (smoothmuscle), etc. In a specific embodiment, a mouse albuminpromoter/enhancer is used which consists of nucleotides 338-668 fromGenBank® accession # U04199, followed by the sequence gagtcgacggatccgg,followed by nucleotides 1762-1846 from accession # J04738, followed bythe nucleotide sequence tgggggtgggggtgggg followed by nucleotides1864-2063 of accession # J04738. In one embodiment, the mousepromoter/enhancer is active in hepatocyte (e.g., human hepatocytes,hepatocyte cultures, etc.) and is useful for tissue-specific expressionin liver tissue. It will also be clear that target-specific promotersnot yet identified can be used to target expression of the presentribozymes to the selected tissue(s). Once a target-specific promoter isidentified its binding sequence can be determined by routine methodssuch as sequence analysis may be used. The promoter is defined bydeletion analysis, mutagenesis, footprinting, gel shifts andtransfection analyses (Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989). Pathogen-specific promoters can be used in the presentnucleic acid constructs.

[0204] 5.4.1. Bacterial Specific Promoters/Expression

[0205] The present invention provides bacterial promoters that allow fortight regulation of transcription and enhanced expression. In oneembodiment, a novel promoter called LEASHI has been constructed fromthree elements (see FIG. 1). The first element, termed RIP is acombination of two consensus sites at −10(TATAAT) and −35(TTGACA)located with respect to transcription initiation. The second element isbased on the lacI repressor binding sequence (termed lac operatorsequence) which is placed between the −10 and −35 consensus sites. Thisis in contrast to the conventional lac and tac promoters where the lacoperator is found downstream of the −10 consensus element. Placement ofthe lac operator between the −10 and −35 sites, more effectively blocksRNA polymerase binding to the promoter, thus enhancing transcriptionalcontrol from the promoter. Thus, the levels of lacI repressor proteinpresent, which binds to the operator sequence and hence determines therate of transcription, are controlled in two ways; 1) by endogenouslyexpressed lacI protein and 2) by a plasmid expressing the lacI gene.Under normal conditions, the lacI repressor protein binds to the lacoperator sequence and prevents transcription by blocking RNA polymerasebinding. The promoter is ‘switched on’ following the addition ofisopropyl B-D-thiogalacto pyranoside, which binds and subsequentlytitrates out the repressor protein. RNA polymerase can then bind to thepromoter and transcription can proceed.

[0206] The third element of the LEASHI promoter is termed the UPelement. The UP element is an adenine/thymine rich sequence which isplaced immediately upstream of the −35 element. Addition of the UPelement, further increases expression from this promoter. Accordingly,the invention provides the use of a LEASHI promoter to express the toxicagents of the invention.

[0207] In a specific embodiment of the invention, the promoter which isoperably linked to a nucleic acid encoding a toxic agent or ribozyme isthe LEASHI promoter.

[0208] In specific embodiment, a ribozymes of the invention is operablylinked to a LEASHI promoter. In another specific embodiment of theinvention, a toxic agent of the invention is operably linked to a LEASHIpromoter.

[0209] In a specific embodiment, the invention encompasses expression ofDicF1 from a ribozyme cassette under the control of a regulatablepromoter, such as the LEASHI promoter.

[0210] In another embodiment of the invention, the lacI operatorsequence of the LEASHI promoter is placed 5′ of the −35 consensus site.In another embodiment of the invention, the lacI operator sequence ofthe LEASHI promoter is placed 3′ of the −10 consensus site. In otherembodiment of the invention, one or more additional lacI operatorsequences are added to the LEASHI promoter and are placed 5′ to the −35consensus site and/or 3′ of the of the −10 consensus site.

[0211] In other specific embodiments, the invention provides for the useof an anr, arc, or proC promoter. Both are transcriptionally off in E.coli and on in Pseudomonas aeruginosa. These promoters provide theadvantage of allowing controlled expression of the toxic agents in thepathogen (Pseudomonas), while allowing the packaging strain (E. coli) tobe protected from the toxic actions of the toxic agent therapeutic. Suchpromoters are particularly useful to facilitate manufacturing of thedelivery vehicle. Such promoters also enable bacterial specifictargeting of the gene therapeutic in the patient. In specificembodiments, an anr promoter is operably linked to a sequence encoding atoxic agent (such as doc, gef chpBK, or kicB, etc), and may be used, forexample, for the eradication of Pseudomonas.

[0212] In other specific embodiments, the invention provides for the useof the TSST-1 promoter. TSST-1 is an environmentally regulatedstaphylococcus-specific promoter. TSST-1 is useful to express doc orother toxic agents. A staphylococcus specific phage capable ofdelivering the transfer plasmid into S. aureus strains is used tospecifically target the Staphylococcal pathogen.

[0213] Other classical bacterial inducible promoters are renowned fortheir inability to tightly control transcription, and a significantlevel of background expression is characteristically observed. Asignificant advantage of the promoter of the present invention is thatit will alleviate the high levels of background commonly observed ininducible promoters. A limiting factor leading to high background levelsof transcription when a promoter of interest is on a high-copy numberplasmid is due to the lack of repressor molecules available to bind tothe promoters. The present invention overcomes this problem by using alacI expression plasmid and secondly, by placement of the lac operatorbetween the −35 and −10 consensus elements which more effectively blockstranscription during normal conditions. Furthermore, the UP elementplaced immediately upstream of the −35 region enhances transcriptionfrom the core promoter.

[0214] The invention also relates to the rrnB promoter. In oneembodiment of the invention, the promoter is the rrnB promoter ismodified such that one or more lacI operator sites are added to thepromoter. An example of such a modified rrnB promoter is shown in FIG.1B. In another embodiment of the invention, the lacI operator sequenceof the rrnB promoter is placed 3′ of the −10 consensus site. In otherembodiment of the invention, one or more additional lacI operatorsequences are added to the rrnB promoter and are placed 5′ to the −35consensus site and/or 3′ of the of the −10 consensus site.

[0215] 5.5. Host Cells

[0216] The present invention encompasses the expression of the toxicagents and/or ribozymes in primary cells, animal, insect, fungal,bacterial, and yeast cells for in vitro screening assay and ex vivo genetherapy. The present invention also encompasses the expression of thetoxic agents and/or ribozymes in cell lines for in vitro screening assayand ex vivo gene therapy. In accordance with the present invention, avariety of primary or secondary cells or cell strains may be usedincluding but not limited to cells isolated from skin, bone marrow,liver, pancreas, kidney, adrenal and neurological tissue to name a few.Other cell types that may be used in accordance with the presentinvention are immune cells (such as T-cells, B-cells, natural killercells, etc.), macrophages/monocytes, adipoctyes, pericytes, fibroblasts,neuronal cells, reticular cells etc. In a further embodiment, secondarycell lines may be used as engineered responsive cells and tissues inaccordance with the present invention, including, but not limited tohepatic cell lines, such as CWSV, NR, Chang liver cells, or other celllines such as CHO, VERO, BHK, Hela, COS, MDCK, 293, 373, HUVEC, CaSkiand W138 cell lines. A toxic agent or ribozyme of the invention may alsobe expressed in any cell line which is not sensitive to the effects ofthe toxic agent or ribozyme (e.g., a cell which is resistant to theparticular toxic agent or ribozyme, or a cell which co-expresses aneutralizing agent or antidote).

[0217] For long term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the selected toxic agent and/or ribozyme may be engineered. Whena toxic agent is to be stably expressed, expression may be controlled byan inducible promoter, or, the cell may be engineered to co-express anantidote to the toxic agent, in order to allow the cell to surviveduring production of a toxic agent. Rather than using expression vectorswhich contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter sequences, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to grow to formfoci which in turn can be cloned and expanded into cell lines. Thismethod may advantageously be used to engineer cell lines. This methodmay advantageously be used to engineer cell lines which express theselected gene products. Such cell lines would be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the selected gene product.

[0218] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes can be employed in tk⁻, hgprt⁻or aprt⁻cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the amninoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

[0219] 5.6. Targets

[0220] The present invention provides a toxic agent or a trans-actingribozyme which targets any cellular, viral, bacterial, fungal, or othersingle cellular or multicellular organism from any known taxonomicfamily, genus, or species. Another embodiment of the invention providesa toxic agent which is lethal or toxic to a pathogen such as a bacteria,fungus, yeast, diseased cell. Such toxic agents may be delivered to thepathogen by the methods of the invention. The microorganisms may be anyvirus, nonvirus, bacterium, or lower eukaryotes such as fungi, yeast,parasites, protozoa, or other eukaryotes that may be consideredpathogens of humans, animals, fish, plants, or other forms of life. Inseveral embodiments, the targets of the antimicrobial ribozymetherapeutics described herein are the RNAs of invading or normal floramicroorganisms. In other embodiments, the targets of the antimicrobialtoxic agent therapeutics described herein include RNAs, proteins, genesand other molecules of invading or normal flora microorganisms. Thus,the invention has important implications in human and veterinarymedicine.

[0221] The toxic agents of the present invention may be engineered totarget essential genes, gene products, or processes necessary for growthof parasites, bacteria, virus life cycles, etc., and expression can bedriven with tissue-specific or pathogen-specific promoters. The toxicagents or trans-acting catalytic ribozymes of the present invention maybe engineered to target a wide variety of cellular RNAs, tumor or cancerassociated with RNAs, bacterial RNAs, parasitic RNA etc. For example,ribozyme targets sites are indicated in Tables 11, 13 and 13 herein. Thetoxic agent or trans-acting ribozyme can be targeted to noncellular RNAsnecessary for growth of parasites, bacteria, virus life cycles, etc.,and expression can be driven with tissue-specific or pathogen-specificpromoters.

[0222] The virion construct used in this method can comprise any nucleicacid encoding a toxic agent or ribozyme, particularly those describedherein targeted to essential genes of the pathogen or diseased cell. Thevirion can be a bacteriophage, or other virus selected for its abilityto target a specific cell-type, microorganism or animal. Thebacteriophage can be lambda, P1, Phi-11 or other phage. When P1 is thevirion, the transfer plasmid can further comprise a PAC site and PAC ACBgenes. This construct is preferred when using P1. Alternatively, thevirion can be selected because it has a broad range of targets.

[0223] Important examples which are specifically presented in theapplication are:

[0224] A) Use of the LEASHI promoter with a Bacterial target (such as E.coli) to direct expression of the toxic agent such as doc, gef, chpBK,kicB or DicF1;

[0225] B) Use of the LEASHI promoter with a Bacterial target (such as E.coli) to direct expression of the toxic agent comprising Sof sense RNA;

[0226] C) Use of the anr, arc, or proC promoter with a Pseudomonastarget (such as P. aeruginosa) to direct expression of a toxic agentsuch as doc, gef, chpBK, kicB or DicF1;

[0227] D) Use of the TSST-1,hla, or SrcB promoter with a Staphylococcustarget (such as S. aureus) to direct expression of the toxic agent suchas doc, gef, chpBK, kicB, pemK, hok, relF, sigB, or lysostaphin;

[0228] E) Use of the albumin promoter with a Hepatitis B virus target(chosen to cleave the viral RNA pregenome, S protein, and polymerase/andx protein transcripts using the same ribozyme target site);

[0229] F) Use of the albumin promoter with Hepatitis B virus andHepatitis C targets (using trans-acting ribozyme target sites on bothHepatitis B virus and Hepatitis C);

[0230] G) Use of generic promoters active in erythrocytes, using aribozyme targeted to highly conserved regions of the EMP-1 proteinfamily from P. falciparum, which are necessary for cytoadherence andantigenic variation in malaria; and

[0231] H) Use of the keratin 7 promoter, with trans-acting ribozymestargeted to a specific sites near the translational start site of the E6protein, a site known to be critical for expression of both the E6 andE7 proteins which are intimately involved in cervical carcinogenesis, aswell as a more 3′ site in a highly conserved region of the E6 protein.

[0232] Examples of bacterial pathogens that can be targeted by a toxicagent or trans-acting ribozyme of the present invention include, but arenot limited to, species of the following genera: Salmonella, Shigella,Chlamydia, Helicobacter, Yersinia, Bordatella, Pseudomonas, Neisseria,Vibrio, Haemophilus, Mycoplasma, Streptomyces, Treponema, Coxiella,Ehrlichia, Brucella, Streptobacillus, Fusospirocheta, Spirilluim,Ureaplasma, Spirochaeta, Mycoplasma, Actinomycetes, Borrelia,Bacteroides, Trichomoras, Branhamella, Pasteurella, Clostridium,Corynebacterium, Listeria, Bacillus, Erysipelothrix, Rhodococcus,Escherichia, Klebsiella, Pseudomonas, Enterobacter, Serratia,Staphylococcus, Streptococcus, Legionella, Mycobacterium, Proteus,Campylobacter, Enterococcus, Acinetobacter, Morganella, Moraxella,Citrobacter, Rickettsia, Rochlimeae, as well as bacterial species suchas: P. aeruginosa; E. coli, P. cepacia, S. epidermis, E. faecalis, S.pneumonias, S. xylosus, S. aureus, N. meningitidis, S. pyogenes,Pasteurella multocida, Treponema pallidum, and P. mirabilis.

[0233] The pathogen of the present invention can also include, but isnot limited to pathogenic fungi such as Cryptococcus neoformans;Blastomyces dermatitidis; Aiellomyces dermatitidis; Histoplasmacapsulatum; Coccidioides immitis; Candida species, including C.albicans, C. tropicalis, C. parapsilosis, C. guilliermondii and C.krusei, Aspergillus species, including A. fumigatus, A. flavus and A.niger, Rhizopus species; Rhizomucor species; Cunninghammella species;Apophysoinyces species, including A. saksenaea, A. mucor and A. absidia;Sporothrix schenckii Paracoccidioides brasiliensis; Pseudallescheriaboydii, Torulopsis glabrata; Trichophyton species, Microsporum speciesand Dermatophyres species, as well as any other yeast or fungus nowknown or later identified to be pathogenic.

[0234] Furthermore, the pathogen of the present invention can be aparasite, including, but not limited to, members of the Apicomplexaphylum such as, for example, Babesia, Toxoplasma, Plasmodium, Eimeria,Isospora, Atoxoplasma, Cystoisospora, Hammondia, Besniotia, Sarcocystis,Frenkelia, Haemoproteus, Leucocytozoon, Theileria, Perkiizsus andGregarina spp.; Pneumocystis carinii; members of the Microspora phylumsuch as, for example, Nosema, Enterocytozoon, Encephalitozoon, Septata,Mrazekia, Amblyospora, Ameson, Glugea, Pleistophora and Microsporidiumspp.; and members of the Ascetospora phylum such as, for example,Haplosporidium spp., as well as species including Plasmodium falciparum,P. vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana,L. tropica, L. major, L. aethiopica, L. donovani, Trypanosoma cruzi, T.brucei, Schistosoma manisoni, S. haematobium, S. japonium; Trichinellaspiralis; Wuchereria bancrofti; Brugia malayli; Entamoeba histolytica;Enterobius vermiculoarus; Taenia solium, T. saginata, Trichomonasvaginatis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidiumparvum; Pneumocytis carinii, Babesia bovis, B. divergens, B. microti,Isospora belli, L hominis; Dientamoeba fragilis; Onchocerca volvulus;Ascaris lumbricoides; Necator americanis; Ancylostoma duodenale;Strongyloides stercoralis; Capillaria philippinensis; Angiostrongyluscantonensis; Hymenolepis nana; Diphyllobothrium latum; Echinococcusgranulosus, E. multilocularis; Paragonimus westerinani, P. caliensis;Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciolahepatica, Sarcoptes scabiei, Pediculus humanus; Phthirlus pubis; andDermatobia hominis, as well as any other parasite now known or lateridentified to be pathogenic.

[0235] Examples of viral pathogens include, but are not limited to,retroviruses (human immunodeficiency viruses), herpes viruses (herpessimplex virus; Epstein Barr virus; varicella zoster virus),orthomyxoviruses (influenza), paramyxoviruses (measles virus; mumpsvirus; respiratory syncytial virus), picoma viruses (Coxsackie viruses;rhinoviruses), hepatitis viruses (hepatitis C), bunyaviruses(hantavirus; Rift Valley fever virus), arenaviruses (Lassa fever virus),flaviviruses (dengue fever virus; yellow fever virus; chikungunyavirus), adenoviruses, birnaviruses, phleboviruses, caliciviruses,hepadnaviruses, orbiviruses, papovaviruses, poxviruses, reoviruses,rotaviruses, rhabdoviruses, parvoviruses, alphaviruses, pestiviruses,rubiviruses, filiviruses, coronaviruses, as well as any virus of thefamily of picomaviridae; caliciviridae; togaviridae; flaviviridae;coronaviridae; rhabdoviridae; filoviridae; paramyxoviridae;orthomyxoviridae; bunyaviridae; arenaviridae; reoviridae; retroviridae;hepadnaviridae; parvoviridae; papovaviridae; adenoviridae;herpesviridae; and poxyviridae, and any other virus now known or lateridentified to be pathogenic.

[0236] 5.7. Target Selection

[0237] One critical component in the development of the therapeutics ofthe invention is the selection of appropriate targets.

[0238] Toxic Agent Targets

[0239] The inventions toxic agents are selected based on their abilityto inhibit the growth of a pathogen or selected cell or cause lethalityin a pathogen or selected cell, or render a pathogen or selected cellless fit. Several specific examples of toxic agents are described hereinwhich serve to illustrate the selection of a toxic agent of theinvention.

[0240] For example, a toxic agent may be an addiction system toxin (suchas doc). Doc encodes a toxin which is translationally coupled to aprotein called phd. Phd is an antidote to doc, and acts to neutralizethe toxic effects of doc. The two proteins, phd and doc form an operonon the P1 plasmid in which phd precedes doc. Further, the phd genecontains a ribosome entry site and is translated efficiently. The nativedoc gene however, lacks a recognizable ribosome entry site and istranslated poorly. Thus, doc was selected because of its potentialtoxicity when expressed in a cell or pathogen lacking the correspondingantidote, phd. In this embodiment, doc has been engineered to beuncoupled from phd. For example, doc is engineered into a separateplasmid from phd. The plasmid containing doc has also been engineeredsuch that a ribosome entry site has been constructed upstream of thenucleic acids encoding doc in order to increase the levels oftranslation of doc. This plasmid is containing the toxic agent and/orribozyme of the invention is called the Transfer plasmid. In onespecific embodiment of the invention, the Transfer plasmid encodes thetoxic agent doc.

[0241] A packaging strain (e.g., bacteria cell) is then used to packagethe Transfer plasmid containing doc into a bacteriophage phage head. Thepackaging strain cells contain the P1 plasmid as well as the Transferplasmid with the uncoupled doc and ribosome entry site. The packagingstrain may also include a third plasmid, if necessary, which encodesadditional phd protein which can act to protect the packaging strainagainst the toxicity of doc (e.g., if the promoter of the Transferplasmid is leaky and leads to production of doc in the packaging cell).

[0242] Thus, the packaging strain acts to package the transfer plasmidcontaining the toxic agent (such as doc) into phage heads or virions.Phage lysates of the packaging strain contain the infectiousbacteriophage virions.

[0243] The phage lysates are then used to infect a selected pathogen(e.g., E. coli, P. aeruginosa, etc.). Further, the phage lysate may beused to prepare a therapeutic of the invention, such as a pharmaceuticalpreparation. Phage may be delivered to a bacteria or pathogen or a hostwith a pathogenic infection by methods described herein, or by anymethod known in the art. For example, the phage lysates may belyophilized and delivered to a host in need of treatment for a bacterialinfection, fungal infection, etc.

[0244] The above targeting method, wherein the virion is a bacteriophageis provided. The bacteriophage can be lambda, P1 or other phage. Thetargeting method, wherein the Transfer plasmid further comprises a PACsite and PAC ABC genes is also provided. The bacteriophage P1 which isengineered to be packaging deficient is also provided. This construct ispreferred when using P1.

[0245] Antisense Targets

[0246] A toxic agent of the invention may be an antisense moleculeselected to target an antidote of a toxic protein, or selected to targetan essential RNA critical to the survival of a pathogen or selectedcell. The proposed target of the toxic antisense molecule of theinvention may also be the RNA of any gene which plays a critical role inthe survival of the pathogen, or which is essential to the pathogen'slife cycle. The present invention also encompasses modifications tonaturally occurring antisense molecules which modulate the expression ofan essential gene product of a pathogen. For example, as describedbelow, one proposed target of an antisense of the invention is the ftsZgene whose gene product plays a critical role in the initiation of celldivision of E. coli. For example, the toxic agent may be an antisensemolecule which is constructed to be modified and enhanced such that isit more homologous to its target RNA. Thus, as in the case of DicF, theantisense sequence has been modified and enhanced to engineer the DicF1antisense toxic agent, which has increased complementarity to its targetRNA. Further, the DicF1 or DicF1-like antisense molecule has enhancedproperties in that it may be expressed and delivered by the methods ofthe invention, thus providing the target cell with increased levels ofthe toxic antisense RNA.

[0247] Third, a toxic agent may be selected to target an essentialantisense molecule. Thus, a toxic agent may be a sense molecule which isdesigned to be complementary to an essential antisense RNA. An exampleof an essential antisense molecule is Sof Sof is an antisense antidotefor the chromosomally encoded toxin called gef (Poulsen, L., et al.,1991, Mol. Microbiology 5:1639-48). Sof normally acts to regulate thelevels of gef in the bacterium. The inventors of the present inventionhave designed sense molecules which are complementary to Sof The sensemolecules against Sof act to inhibit the ability of Sof to regulate gef,and thus cause toxicity in the pathogen by allowing the endogenous geflevels to become toxic.

[0248] Ribozyme Targets

[0249] For ribozymes to be effective anti-microbial therapy, it ispreferable to target the RNA of, for example, several key proteins,tRNA, rRNA or any other RNA molecule essential for cell viability orfitness, in order to insure complete inactivation and prevent escape ofthe invading microorganism.

[0250] The complexity of human RNA is about 100 fold lower than that forhuman DNA, and specificity can be achieved with as few as 12-15 basepairs. The stability of the RNA-RNA duplex is effected by severalfactors, such as GC content, temperature, pH, ionic concentration, andstructure. The nearest neighbor rules can provide a useful estimate ofthe stability of the duplex (Castanotto et al. “Antisense Catalytic RNAsas Therapeutic Agents” Advances in Pharmacol. 25:289-317, 1994).

[0251] The catalytic ribozyme of the invention also includes a catalyticsequence, which cleaves the target RNA near the middle of the site towhich the target RNA-specific sequences bind. In the hammerhead-type ofribozyme, the catalytic sequence is generally highly conserved. Theconserved catalytic core residues are 5′ CUGANGA3′ and 5′ GAAA 3′ linkedby an evolutionarily conserved stem-loop structure.

[0252] The most conserved and probably most efficiently cleaved sequenceon the target RNA is 5′ GUC 3′. However, NUX (wherein X=A, U or C) canalso be cleaved efficiently. Such cleavage sites are ubiquitous in mostRNAs allowing essentially all RNA's to be targeted (Whitton, J. Lindsay“Antisense Treatment of Viral Infection” Adv. in Virus Res. Vol. 44,1994).

[0253] With regard to the selection of the appropriate sites on targetRNA, it is known that target site secondary structure can have an effecton cleavage in vitro (Whitton, 1994 supra). A number of procedures areavailable to select accessible sites in RNA targets. In a preferredprocedure, a library screen may be employed to select appropriate siteson the target RNA. Accessibility of the selected site may then beconfirmed using techniques known to those skilled in the art. Thus, theselected target molecule's sequence can be routinely screened forpotential secondary structure, using the program RNAFOLD (from thePCGENE group of programs or available on the Internet). Thus, reasonablepredictions of target accessibility can be made. Computer assisted RNAfolding (Castanotto et al., 1994), along with computational analysis for3-dimensional modeling of RNA (Major et al., Science 253:1255-1260, 1991and Castanotto et al., 1994) is certainly effective in guiding thechoice of cleavage sites.

[0254] The nucleic acid, wherein at least one trans-acting ribozyme istargeted to a ccdA, kis, pemI, parD, phd, higa, chpAI, chpBI, kicA, soc,sos, srnC, flmB, pndB, sof korA, korB, korC, korD, korE, or korFtranscript of the pathogen is provided. The nucleic acid, wherein atleast one trans-acting ribozyme is targeted to the rpoA transcript ofthe pathogen is provided. The nucleic acid, wherein at least onetrans-acting ribozyme is targeted to the secA transcript of the pathogenis provided. The nucleic acid, wherein at least one transacting ribozymeis directed to the dnaG transcript of the pathogen is provided. Thenucleic acid, wherein at least one trans-acting ribozyme is directed tothe ftsZ transcript of the pathogen is provided. A nucleic acid encodinga multi-ribozyme can encode all or some of the above trans-actingribozymes. The ribozymes can all be under the control of a singlepromoter.

[0255] For example, several bacterial genes, essential for viability andunrelated in activity, have been selected and are described herein tohighlight how the selection of appropriate mRNA targets is carried outfor the preferred construction of the antimicrobial agent againstprokaryotic targets. Cross-genera RNA targets can be used to design anantimicrobial that can have broad application, modified by thespecificity of the promoter. In addition, several toxic agents aredescribed herein to highlight how the selection of appropriate toxicagents is carried out for the preferred construction of theantimicrobial agent against prokaryotic targets.

[0256] In one embodiment of the invention, the first ribozyme targets anessential transcription factor, the second ribozyme targets an essentialgeneral secretory component, the third ribozyme targets an essentialcomponent of the primosome required for DNA biosynthesis and the fourthribozyme targets an enzyme required for cell division. Consequently, theribozymes are redundant in the fact that they inhibit growth byspecifically targeting a fundamental process required for bacterialgrowth. Thus, this can minimize the development of resistance to theantimicrobial therapeutic.

[0257] For example, one target is the essential protein, rpoA or thealpha subunit of RNA core polymerase. rpoA was selected rather than theother components of the RNA polymerase holoenzyme, because it is thoughtto facilitate the assembly of an active RNA polymerase enzyme complex.Inactivation of the rpoA transcript results in a decrease in theintracellular concentration of the holoenzyme RNA polymerase renderingthe cell less able to respond to changes demanded of it once it hasinvaded a new host. The nucleotide sequence of rpoA is known for a largenumber of microorganisms (>20 genera) and they are readily availablefrom GenBank®.

[0258] A second example of a ribozyme target can be the mRNA of the secagene from bacteria. The product of this gene is the essential andrate-limiting component of the general secretory pathway in bacteria(Bassford et al., 1994, Nucleic Acids Reseaarch Apr. 11, 22(7):1326;Nucleic Acid Research. 22(3):293-300). SecA has been found in everyprokaryotic cell investigated to date. Additionally, its biosynthesis istranslationally coupled to the upstream gene, X (Schmidt et al., 1991,J. Bacteriol. 173(20):6605-11), presenting a convenient target for aribozyme. Inhibition or decreased synthesis of seca is also sufficientto confer a reduction in viability to the cell (Schmidt et al., 1987, J.Bacteriol. 171(2):643-9). Furthermore, as a pathogen responds to changesrequired of the infectious process a change in the availability of a keyprotein such as seca will disadvantage the pathogen enabling the host tocounteract it. Finally, control over the secretion-responsive expressionof seca is at the level of translation (Christoffersen et al., 1995, J.Med. Chem. 38(12):2023-37), and the regulatory sequences within itspolycistronic message have been localized to a region comprised of theend of the upstream gene, X, and the beginning of secA. Consequently,inactivation of the transcript by the catalytic cleavage of a ribozymehas profound consequences for the viability of the invadingmicroorganism.

[0259] The third ribozyme can target essential factor for DNAbiosynthesis, such as DnaG. Every 1 to 2 seconds, at least 1,000 timesfor each replication fork within E. coli, priming of an Okazaki fragmentis repeated as a result of an interaction between the cellular primasednaG (Bouche et al., 1975, J. Biol. Chem. 250:5995-6001) and dnaB(Marians, K. J. 1996, Replication Fork Propagation, p. 749-763. In F.C.Neidhardt (ed.), Escherichia coli and Salmonella: Cellular and MolecularBiology, 2nd ed, vol. 1. American Society for Microbiology, Washington,D.C.). As would be expected of a protein required every 1 to 2 secondsduring replication, a lesion within DnaG or an alteration in itsconcentration results in an immediate stop phenotype (Marians, K. J.1996, Replication Fork Propagation, p. 749-763. In F. C. Neidhardt(ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology,2nd ed, vol. 1. American Society for Microbiology, Washington, D.C.);Weschler et al., 1971, Mol. Gen. Genet. 113:273-284). Therefore,inactivation of the DnaG message by a ribozyme should have profoundcellular consequences in that general priming of the lagging strand isreduced if not eliminated. DnaG is a component of the primosome, amulti-protein complex responsible for priming replication. Any of thecomponents of the primosome, either individually or in any combination,can serve as a target for inactivation of the primosome and, thus, killthe cell. The other components of the primosome are DnaB, DnaC, DnaT,PriA, PriB, and PriC. Thus, the primosome is also sufficiently complexto provide numerous other targets (DnaB, DnaC, DnaT, PriA, PriB andPriC) for inactivation by the trans-acting ribozyme.

[0260] A fourth target can be ftsZ. This gene also encodes an essentialprotein, ftsZ, that is required for cell division in that it isresponsible for the initiation of separation. ftsZ was selected becausecleavage of the ftsZ RNA leads to inhibition of cell division and areduction in viability. Any toxic agent or ribozyme which targets ftsZ(such as DicF1) may be used to inhibit division of a cell requiring theftsZ gene product. Also, for example, upon cleavage of the ftsZ messageby a ribozyme, such ribozyme can attack additional copies of the ftsZmessage inhibiting the division of the cell. The nucleotide sequence offtsZ like the other targets selected, is commonly available fromGenBank®.

[0261] It should be clear that any other essential protein of a pathogencan have its message targeted in the present invention, and thatdetermining which proteins are essential can be routinely determinedaccording to standard protocols in the art. In fact, there are over52,000 viral, 41,000 bacterial and 12,300 fungal sequences deposited inthe public section of the Entrez Database at the National Center forBiotechnology Information. Any of these can be used to design thecatalytic trans-acting ribozyme of the invention.

[0262] In addition to targeting mRNA of essential proteins, ribozymesmay be targeted against other RNA species within the cell. Specifically,appropriate targets in bacteria, fungi and other lower eukarytoesinclude ribosomal RNA such as Small Subunit RNAs (SSU) or Large Subunit(LSU) and tRNA molecules required for protein synthesis. With respect topathogenic Staphylococci, the RNA III moiety in a relatively lowabundance transcript which is not translated and should be accessiblefor cleavage. As long as the RNA targeted contains a canonical ribozymecleavage domain, the ribozyme therapeutic can hybridize and cleave thecomplementary RNA, thus impacting the fitness of the microbial cell.Additionally, over 3000 rRNA species have been sequenced and aligned.This information is available from the Ribosomal Database Project andshould facilitate rapid design and adaptation of ribozyme(s) againstsuch targets. For example the 16S rRNA molecule of bacteria isespecially attractive in that there are over 4000 copies of the 16S rRNAper cell. Consequently, a reduction in number slows the process ofprotein synthesis in so far as the 16S rRNA molecule is involved in theprocess of translational initiation. Thus, a toxic agent or ribozymedirected against mRNA and rRNA impacts the fitness of the offendingmicroorganism.

[0263] 5.8. Protection of Toxic Agent and/or Ribozyme Producing Cells

[0264] The nucleic acids coding for the toxic agents or ribozymes can betoxic to the cells that are needed to produce the toxic agent orribozyme-carrying virions. When using a broad host-range virus like P1,the organism used to produce the virion can be different from the targetorganism. In this way, the producing strain is resistant to the toxiceffects of the toxic agents or ribozymes because they are notefficiently expressed in the producing strain, due to species-specificpromoter elements, and the ribozymes will not have any target RNAmolecules to attack, due to the species-specific sequences that targetthe ribozymes. When using a species-specific virus that must beexpressed and assembled within a strain of the targeted microorganism,this toxicity becomes a significant concern. The assembly of a virionconsisting of anti-E. coli ribozyme or toxic agent genes packaged inlambda will illustrate the approach used to circumvent the toxicity. Forexample, the ribozymes directed against RNA species of E. coli isexpressed from an artificial promoter containing consensus promoterelements. This promoter provides high level transcription of theribozyme immediately upon infection of targeted cells. In order toprevent the unwanted death of the producing strain of E. coli,transcription is repressed in the producing strain by a mechanism notavailable to the wild type strains that are targeted for killing.Sequences constituting the DNA binding sites for a heterologoustranscription factor are interspersed between the essential activatingelements of the ribozyme promoter. Expression of the heterologoustranscription factor in the producing strain results in the occlusion ofthe activating promoter elements and preventing the binding of RNApolymerase. As an example, the gene for the Saccharomyces cerevisiaetranscription factor Ste12p may be expressed in E. coli and bind to itsbinding sites, the pheromone response element, located within theribozyme promoter. Ste12p will not be found in wild strains of E. coli;therefore, the ribozyme promoter will be accessible to RNA polymerasefollowing delivery of the plasmid to the targeted cells.

[0265] An alternative strategy that can protect the producing strainfrom the toxicity of the ribozymes employs ribozyme-resistant versionsof the targeted RNA molecules. This strategy can be used when the targetRNA molecule codes for a protein. The ribozyme target site within themRNA molecule is mutated by site-directed mutagenesis such that theamino acid sequence of the translated protein does not change but themRNA sequence no longer serves as a substrate for the ribozyme. Forexample, hammerhead ribozymes require an NUX sequence within the targetmRNA for cleavage to occur. By changing this sequence to something else,the ribozyme will not cleave the mRNA. This type of ribozyme resistantversion of the target RNA can be expressed from a plasmid or integratedinto the chromosome of the producing strain and thus render this strainresistant to the toxic effects of the ribozyme.

[0266] Another strategy that can protect the producing cell from thetoxicity of a toxic agent employs co-expression of a neutralizing agentor antidote. Such co-expression of an antidote or neutralization agentprotects the packaging cell from the toxic effects of the encoded toxicagent. Such a strategy is particularly useful is the promoter used toexpress the toxic agent is leaky, and leads to expression of the toxicagent in the producing cell. For example, a packaging strain (e.g.,bacteria cell) may used to package the a viral vector containing a toxicagent into a bacteriophage phage head. Survival of the packaging cell oroptimization of the quantities of vector or phage made by the producing.cell may require co-expression of an antidote or neutralization agent inthe producing cell. A neutralization agent is any molecule (such asprotein, antisense, sense, or other molecule (such as a drug, chemical,etc.)) which counteracts the toxic effects of a toxic agent. By way ofillustration, in a specific example, the packaging strain cells containa bacteriophage P1 plasmid as well as the Transfer plasmid comprisingthe toxic agent doc and a ribosome entry site. In the case that theTransfer plasmid is determined to be toxic to the packaging strain, athird plasmid may be introduced, which encodes an antidote to doc, suchas the phd protein. The additional plasmid with the antidote acts toprotect the packaging strain against the toxicity of doc.

[0267] The improvement in the present invention is that anon-replicative delivery system has an advantage in that once the phagecoat has injected the nucleic acid into the targeted bacterium, theexpression of the toxic agent or ribozyme will destroy the rnicrobe, asopposed to a lytic infection cycle typical of an intact bacteriophage.Consequently, amplification of the phage coat will not be an issue andit is less likely that the non-replicative phage delivery system willgenerate an immune response such that subsequent use of the deliverysystem would be jeopardized. Moreover, if the patient has been exposedto a resistant pathogenic microbe and the therapeutic of the inventionis effective and neutralizes the invading microbe, then it is expectedthat the microbial antigens liberated as a result of the action of thetherapeutic, will illicit sufficient humoral immunity and cell-mediatedimmunity to confer protection against subsequent attacks.

[0268] 5.9. Therapeutics and Pharmaceutical Preparations/Formulationsand Methods for Administration

[0269] The present invention further encompasses the use of a toxicagent and/or ribozymes of the present invention for the treatment ofdisease, viral infection, parasitic infection and microbial infection.The present invention further provides a method of treating a subjecthaving a proliferative disease of a specific tissue by inhibiting cellproliferation in the tissue, comprising administering to the subject atoxic agent and/or ribozyme operably linked to a tissue-specificpromoter sequence, which promoter is specific for the diseased tissue,and whereby the ribozyme and/or toxic agent encoded by the nucleic acidis expressed, cell proliferation is inhibited, and the proliferativedisease is treated.

[0270] The present invention further provides a method of treating asubject having a pathogenic infection or disease, by inhibitingreplication of the pathogen, comprising administering to the subject atoxic agent and/or ribozyme operably linked to a pathogen-specificpromoter, whereby the ribozyme and/or toxic agent encoded by the nucleicacid is expressed, the pathogen is inhibited from replicating or iskilled or rendered less fit, and the infection or disease is treated.The present invention encompasses the toxic agent(s) and/or ribozyme(s)of the present invention in pharmaceutical formulations.

[0271] In several embodiments of the invention, toxic agents orribozymes of the invention are particularly suited as antimicrobialtherapeutics. For example, upon nucleic acid hybridization with thetarget RNA transcript, a ribozyme-RNA complex achieves a catalytic formthat acts as a nuclease to cleave the targeted RNAs. Thus, cleavagedeprives the invading microorganism of essential cellular processeswhich then kills or renders it less fit. Additionally, a toxic agent ofthe invention may also be used as an antimicrobial therapeutic. A toxicagent may be used alone, or in combination with one or more other toxicagents. Thus, delivery of a toxic agent to an invading microorganism,kills or render it less fit. A toxic agent may also be used incombination with one or more ribozymes. Further, a combination ofribozymes and toxic agents may be used as an antimicrobial therapeutic.

[0272] The invention provides use of one or more ribozymes and/or toxicagents directed towards essential, housekeeping, or virulence genes ofone or a series of candidate microorganisms. Inactivation of essentialproteins and virulence determinants render the invading microbesinactive or slow their growth, while at the same time, the essentialprocesses of the host are not significantly affected.

[0273] A method of delivering a toxic agent or ribozyme to a target(e.g., a pathogen) in a subject is provided, comprising a) generating aliposome comprising a promoter and a sequence encoding a toxic agent orribozyme; and b) delivering the liposome to the subject, whereby thetarget-specific promoter directs transcription of the toxic agent orribozyme in the cells of the target. The target can be a pathogen, forexample, a bacteria, fungus, yeast, parasite, virus or non-viralpathogen.

[0274] A method of targeted delivery of a toxic agent or ribozyme to apathogen in a subject is provided, comprising a) generating a virioncomprising non-viral DNA of the invention; b) combining it with aliposome; and b) delivering the liposome containing the virion to thesubject, whereby liposome enters the eukaryotic cell and releases thevirion, which delivers the DNA to the pathogen, whereby thepathogen-specific promoter directs transcription of the toxic agent orribozyme in the cells of the pathogen.

[0275] A method of treating an infection in a subject is provided,comprising administering to the subject the liposome comprising DNAcomprising a target-specific promoter and a sequence encoding a toxicagent or ribozyme, whereby the toxic agent or ribozyme encoded by theDNA is expressed and the infectious agent is killed or weakened. Theliposome used in this method can comprise any ribozyme-encoding nucleicacid, or any toxic agent-encoding nucleic acid, particularly thosedescribed herein targeted to genes of the pathogen. The infection can bebacterial, fungal, yeast, parasitic, viral or non-viral.

[0276] Direct in vivo gene transfer may be carried out with formulationsof DNA trapped in liposomes (Ledley et al., 1987), or in proteoliposomesthat contain viral envelope receptor proteins (Nicolau et al., 1983),and with DNA coupled to a polylysine-glycoprotein carrier complex. Inaddition, “gene guns” have been used for gene delivery into cells(Australian Patent No. 9068389). Lastly, naked DNA, or DNA associatedwith liposomes, can be formulated in liquid carrier solutions forinjection into interstitial spaces for transfer of DNA into cells(WO90/11092). Asialofetuin-labeled liposomes are known to selectivelytarget hepatocytes via the asialoglycoprotein receptor (Wu et al., 1998,Hepatology 27/3:772-8; Hara et al., 1996, Biochim. Biophys. Acta1278/1:51-8; Hara et al., 1995, Gene Therapy 2/10:784-8; Hara et al.,1995, Gene 159/2:167-74; each of which is hereby incorporated byreference in its entirety). Asialoglycoprotein receptor-mediatedendocytosis has been used as a means to effect gene transfer ortransfection using asialofetuin-labeled liposomes charged with a nucleicacid of interest. Other modifications of liposomes, such as thoseemploying sugars or asialoglycans are known.

[0277] Accordingly, in one embodiment of the instant invention,asialofetuin-labeled liposomes are used as non-viral vectors for thedelivery of the DNAzymes, antisense oligonucleotides or ribozymes of theinstant invention. Targeted delivery of the DNAzymes, antisenseoligonucleotides or ribozymes of the instant invention to animal livercells either in vivo or in vitro is thereby achieved.

[0278] Ex vivo gene therapy, wherein target cells are removed from thebody, transfected or infected with vectors carrying designed nucleicacids of the invention, and re-implanted into the body is also provided.Techniques currently used to transfer DNA in vitro into cells includecalcium phosphate-DNA precipitation, DEAE-Dextran transfection,electroporation, liposome- mediated DNA transfer, cationic lipidmediated transfection, such as, for example, Lipofectamine™ ortransduction with recombinant viral vectors (see generally Ausubel etal., 2001, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., which is incorporated by reference in its entirety). Thesetransfection protocols have been used to transfer DNA into a variety ofdifferent cell types including epithelial cells (U.S. Pat. No.4,868,116; Morgan et al., 1987), endothelial cells (WO89/05345),hepatocytes (Ledley et al., 1987; Wilson et al., 1990) fibroblasts(Rosenberg et al., 1988; U.S. Pat. No. 4,963,489), lymphocytes (U.S.Pat. No. 5,399,346; Blaese et al., 1995) and hematopoietic stem cells(Lim et al., 1989; U.S. Pat. No. 5,399,346).

[0279] Viral vectors are often the most efficient gene therapy deliverysystem, and a number of recombinant, replication-defective viral vectorsare well known in the art to transduce (i.e., infect) cells both ex vivoand in vivo. Such vectors include retrovirus, adenovirus,adeno-associated virus, baculovirus and herpesvirus vectors.

[0280] Parenteral administration, if used, is generally characterized byinjection (intravenous, intradermal, subcutaneous and intramuscular).Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution of orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system such that a constant level ofdosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which isincorporated by reference herein. In certain preferred embodiments ofthe invention administration is parenteral.

[0281] The present invention relates to prophylactic administration. Forexample, many hospital patients or immunocompromised hosts areparticularly susceptible to pathogenic infections. Further, manyhospital strains of pathogens are resistant to traditional antibiotictreatment, such as Penicillin. The therapeutics of the invention areparticularly useful for preventing pathogenic infection or treatinginfections caused by resistant strains of pathogens.

[0282] Suitable carriers for parenteral administration of the substancein a sterile solution or suspension can include sterile saline that cancontain additives, such as ethyl oleate or isopropyl myristate, and canbe injected, for example, intravenously, as well as into subcutaneous orintramuscular tissues.

[0283] Topical administration can be by creams, gels, suppositories,aerosols, sprays, and the like. Ex vivo (extracorporeal) delivery can beas typically used in other contexts. In a preferred therapeutic use, theDNAzymes, antisense oligonucleotides, and/or ribozymes of the inventionare administered to a subject with one or more papillomavirus infectionssuch as warts of the hands, warts of the feet, warts of the larynx,condylomata acuminata, epidermodysplasia verruciformis, flat cervicalwarts, cervical intraepithelial neoplasia, or any other infectioninvolving a papillomavirus. It is generally preferred to apply thetherapeutic agent in accordance with this invention topically orinterlesionally. Other forms of administration, such as transdermal orintramuscular administration may also be useful. Inclusion in ointments,salves, gels, creams, lotions, sprays, inhalants or suppositories ispresently believed to be highly useful. The DNAzymes, antisenseoligonucleotides, and/or ribozymes of the invention may also be used inprophylaxis. Such may be accomplished, for example, by providing themedicament as a coating in condoms or in a spermicidal formulation foruse alone or in conjunction with condoms, diaphragms, and the like. Inpreferred embodiments of the invention, administration is as a topicaltreatment. In one embodiment of the invention, treatment of infectionsassociated with bums or open wounds, topical administration may bepreferred.

[0284] Oral administration is also provided. Suitable carriers for oraladministration include one or more substances which can also act asflavoring agents, lubricants, suspending agents, or as protectants.Suitable solid carriers include calcium phosphate, calcium carbonate,magnesium stearate, sugars, starch, gelatin, cellulose,carboxypolymethylene, or cyclodextrans. Suitable liquid carriers can bewater, pyrogen free saline, pharmaceutically accepted oils, or a mixtureof any of these. The liquid can also contain other suitablepharmaceutical additions such as buffers, preservatives, flavoringagents, viscosity or osmo-regulators, stabilizers or suspending agents.Examples of suitable liquid carriers include water with or withoutvarious additives, including carboxypolymethylene as a pH-regulated gel.

[0285] The therapeutics of the invention can be administered to asubject in amounts sufficient to produce an antibiotic effect or toinhibit or reduce the activity of the target pathogen. Optimal dosagesused will vary according to the individual, on the basis of age, size,weight, condition, etc., as well as the particular modulating effectbeing induced. One skilled in the art will realize that dosages are bestoptimized by the practicing physician and methods determining dosage aredescribed, for example, in Remington's Pharmaceutical Sciences (Martin,E. W., ed., Remington's Pharmaceutical Sciences, latest edition, MackPublishing Co., Easton, Pennsylvania). Treatment can be at intervals andcan be continued for an indefinite period of time, as indicated bymonitoring of the signs, symptoms and clinical parameters associatedwith a particular infection. The parameters associated with infectionare well known for many pathogens and can be routinely assessed duringthe course treatment.

[0286] In one preferred embodiment, the present invention providescompositions containing one or more nucleic acids of the invention thatmay be used to treat viral infections. While individual needs vary, adetermination of optimal ranges of effective amounts of each componentin the composition is within the purview of the skilled artisan. Typicaldosages comprise 0.001 to 100 mg/kg body weight. The preferred dosagescomprise 0.1 to 10 mg/kg body weight. The most preferred dosagescomprise 0.1 to 1 mg/kg body weight. Preferred topical applications willbe in a volume sufficient to cover the papilloma, or sufficient to coverany cervical lesions identified by culposcopy. Topically-applied DNAzmesor antisense oligonucleotides are contemplated to be in the range of 1μg to 250 μg, or preferably in the range of 50 μg to 100 μg, perapplication. For example, a typical HPV topical application is 50 μg in50 μl. For HBV reagents of the invention, total dosage is contemplatedto be in the range of 500 μg to 1000 μg. A determination of optimumdosage ranges will include consideration of toxicity studies and is wellwithin the purview of the skilled artisan.

[0287] In addition to the pharmacologically active agent, thecompositions of the invention may contain pharmaceutically-acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedfor delivery to the site of action.

[0288] Suitable formulations for parenteral administration includeaqueous solutions of the active agents in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers. Liposomes canalso be used to encapsulate an agent for delivery into the cell.

[0289] As described above, pharmaceutical compositions for systemicadministration according to the invention may be formulated for enteral,parenteral or topical administration. Indeed, all three types offormulations may be used simultaneously to achieve systemicadministration of the active ingredient.

[0290] Suitable formulations for oral administration include hard orsoft gelatin capsules, pills, tablets, including coated tablets,elixirs, suspensions, syrups or inhalations and controlled release formsthereof.

6. EXAMPLE: CONSTRUCTION & CHARACTERIZATION OF PROMOTERS

[0291] Classical bacterial inducible promoters are renowned for theirinability to tightly control transcription, and a significant level ofbackground expression is characteristically observed.

[0292] The Leashi Promoter

[0293] The present invention provides bacterial promoters that allow fortight regulation of transcription and enhanced expression. A novelpromoter called LEASHI has been constructed from three elements (seeFIG. 1A). The first element, termed RIP was a combination of twoconsensus sites at −10(TATAAT) and −35(TTGACA) located with respect totranscription initiation. The second element was based on the lacIrepressor binding sequence (termed lac operator sequence) which wasplaced between the −10 and −35 consensus sites. Placement of the lacoperator between the −10 and −35 sites, more effectively blocked RNApolymerase binding to the promoter, thus enhancing transcriptionalcontrol from the promoter. The promoter was designed such that it was‘switched on’ following the addition of isopropyl B-D-thiogalactopyranoside, which binds and subsequently titrates out the repressorprotein. RNA polymerase can then bind to the promoter and transcriptioncan proceed.

[0294] The third element of the LEASHI promoter was the UP element. TheUP element was an adenine/thymine rich sequence which was placedimmediately upstream of the −35 element. Addition of the UP element,further increased expression from this promoter.

[0295] The LEASHI promoter sequence: (SEQ ID NO:1) 5′GATCCTCAGAAAATTATTTTAAATTTCCAATTGACATT GTGAGCGGATAACAATATAATGTGTGGA3′

[0296] A significant advantage of the LEASHI promoter of the presentinvention is that it alleviates the high levels of background commonlyobserved in inducible promoters. A limiting factor leading to highbackground levels of transcription when a promoter of interest islocated on a high-copy number plasmid, is due to the lack of repressormolecules available to bind to the promoters. The present inventionovercomes this problem by using a lacI expression plasmid and secondly,by placement of the lac operator between the −35 and −10 consensuselements which more effectively blocks transcription during normalconditions. Furthermore, the UP element placed immediately upstream ofthe −35 region enhanced transcription from the promoter.

[0297] The LEASH1 promoter (FIG. 1B) was designed as a lacI-regulatedpromoter which a broad spectrum promoter activity in a wide variety ofbacteria. The IPTG inducible LEASH1 functions in Escherichia coli and istightly regulated. It is active in both Gram-negative and Gram-positivebacteria.

[0298] As described herein, ribozymes of the invention have beenoperably linked to the LEASHI promoter. In another specific embodimentof the invention, a toxic agent of the invention was operably linked toa LEASHI promoter.

[0299] The Modified rrnB Promoter

[0300] A novel promoter called the modified rrnB has been constructed(see FIG. 1C). Modified rrnB promoter sequence: (SEQ ID NO:2)5′AGAAAGCAAAAATAAATGCTTGACACTGTAGCGGGAAGGCGTATAATGGAATTGTGAGCGGATAACAATTCACA 3′

[0301] The Anr, Arc, and Proc Promoters

[0302] The Anr (FIG. 1D), proC (FIG. 1E) and Arc (FIG. 1F) promoters arespecies-specific. Both anr and proC are transcriptionally off in E. coliand on in Pseudomonas aeruginosa. These promoters provide the advantageof allowing controlled expression of the toxic agents in the pathogen(Pseudomonas), while allowing the packaging strain (E. coli)to beprotected from the toxic actions of the therapeutic. Such promoters areparticularly useful to facilitate manufacturing of the delivery vehicle.Such promoters also enable bacterial specific targeting of the genetherapeutic in the patient. Pseudomonas aeruginosa ‘specific’ promoters(5′ to 3′) ANR promoter 5′ACTGGCGGATCATCTTCACCATCGGCCGCAACTCCTGCGGGATATCCTCGTC (SEQ ID NO:3)CTCCTCCTCGACCGGCACCCCCATGGTAGCGGCCAGCTCGCGCCCTGCCTGGGAAAGCTGTACATGCTGATCGGCGGCGTCGGTGCCGGCGGCCGGGTCTTCCGCCTGCTCGGCGGTGCCGGTCCGTGCGGCCTTGGCGTCCGCGGCGGCGCGCGATGAGGGCGGCACCTGGGTGGTGATCCAGCCACTGAGGGTCAACATTCCAGTCACTCCGGGAAAAATGGAATTCTTCCATTGGATCGGCCCACGCGTGGCGAACTTGAGCCCCCTTTTCGTCGCCCCTTGACAGGGTGCGACAGGTAGTCGCAGTTGTTTGACGCAAGTCACTGATTGGAAACGCCATCGGCCTGTCAGAAATGGTCGTTGCCAGACCTATGGCTGGCACCCGCATCGCGGCTGCGTTACCCTTACTCCTGTTGTGCCTTTAACCTAG CAAGGAC ProCpromoter 5′ AATTCCTCGAAGTCCTTGCGCTGCTTGTCGTTCATGATGTCGTAGATCAGCGC (SEQID NO:4) ATGCACCTGCTTGTGTTCCAGCGGTGGCAGGTTGATCCGGCGTACATCGCCATCCACCCGGATCATGGGTGGCAGGCCGGCGGAGAGGTGCAGGTCCGAAGCGCCCTGTTTGGCACTGAAGGCGAGCAGCTCGGTAATATCCATGGGACTCCCCAATTACAAGCAAGCAGGTAGAATGCCGCCAAAGCCGCCGTCTCGGACAAGGAAAACACCGGATGAGCCAGGGTGCTTCCAGGACACGCGTGGTGTCCTGCGCCAGACGCGGAACCTCGACACTGGAACAGGAAGATGGCCATCGAGGCCGGCGGTTTCGAGGGCGTCGAGCCGACGCCGACCGCACTTCCATAGGGCGCAGGTAATGTCCACGATAGCAGAGAATATTGCAAAGGTTGCCGCGCGCATCCGTGAGGCAGCGCAAGCTGCGGGGCGCGATCCGGCCACGGTCGGCCTGCTCGCCGTGAGCAAGACCAAGCCCGCCGCCGCGGTGCGCGAGGCGCACGCCGCCGGCCTTCGCGACTTCGGCGAAAACTACCTGCAGGAGGCCCTCGGCAAGCAGGCCGAACTGGCCGACCTGCCCTTGAACTGGCACTTCATCGGCCCCATCCAGTCGAACAAGACGCGGCCCATCGCCGAGCATTTCCAGTGGGTGCACTCGGTGGACCGGTTGAAGATCGCGCAGCGCCTGTCGGAGCAACGCCCGGCCGGGCTGCCGCCCCTGAATGTCTGCCTGCAGGTCAACGTCAGCGGCGAAGCCAGCAAGTCCGGCTGCGCCCCCGAGGACCTGCCGGCCCTGGCCGAGGCCGTGAAGCAACTGCCCAACCTCCGATTGCGTGGCCTGATGGCCATCCCCGAACCCACCGCCGAACGCGCCGCGCAACACGCCGCGTTCGCCCGCCTGCGCGAACTGCTGCTGGACCTGAACCTTGGCCTGGACACCCTGTCCATGGGCATGAGCGACGACCTCGAGGCAGCCATCGGCGAAGGTGCGACCTGGGTCCGCATCGGTACCGCCCTGTTCGGCGCCCGCGACTACGGCGCGCCGGCTTCTTGAATGAATCCC ARC promoter 5′ CTA GAGCTA TTG ATG TGG ATC AAC ATT GTC CAC TAG CCG CTG (SEQ ID NO:5) CCG CCTAAT CTC CAG AAT TGT GAG

[0303] Anr, Arc, and proC promoters, which were expressed preferentiallyin P. aeruginosa, have been isolated and shown to express a toxic agentspecifically in this pathogenic bacterium (See Tables 1 and 2 and FIG.2). Specifically, as shown in Table 1, promoters were cloned upstream ofthe β-lactamase reporter gene in a cassette flanked by multipletranscription terminators. Constructs were transformed into E. coli orP. aeruginosa and plated onto agar containing different amounts ofcarbenicillin. Three repeat evaluations gave the same result. TABLE 1Evaluation of Promoters Utilizing β-Lactamase as a Reporter Gene E. coliP. aeruginosa 25 μg/ml 50 μg/ml 5 mg/ml Construct carbenicillinKanamycin carbenicillin empty vector − + − (no promoter) UPRIPβ-lactamase + + + proC β-lactamase − + + anr β-lactamase − + +

[0304] As shown in Table 2, the chpBK gene was cloned in bothorientations under the control of P. aeruginosa promoters proC and anr.Equal quantities of DNA (500 ng) were transformed into E. coli and P.aeruginosa and plated on agar. Mock transformations were also performedwith ‘no DNA’. +indicates greater than 100 colonies, −indicates nodetectable colonies. Parentheses indicate orientation of the chpBK genein relation to the promoter. Experiments were repeated at least twotimes with the same result. Importantly, plasmids using proC and anr toregulate chpBK expression did not induce cell death in E. coliindicating lack of transcriptional activation function. TABLE 2Evaluation of Promoters that are Expressed Preferentially in P.aeruginosa Construct E. coli P. aeruginosa patent vector + + anr chpBK(positive) + − anr chpBK (negative) + + proC chpBK (positive) + − proCchpBK (negative) + +

[0305] The development of species-specific promoters is particularlyimportant in aspects of the invention in which it is desired to allowindigenous commensal bacteria to be protected from the toxic agents ofthe invention while targeting the pathogenic P. aeruginosa.

[0306] TSST-1 Promoter

[0307] The environmentally regulated staphylococcus-specific promoterTSST-1 has been obtained and a transfer plasmid utilizing this promoteris used to express doc or other toxic agents. A staphylococcus specificphage capable of delivering the transfer plasmid into S. aureus strainsis used to specifically target the Staphylococcal pathogen.

[0308] TSST-I promoter (SEQ ID NO:6) (GenBank® accession number U93688,see also Lindsay, J. A., et al., 1998, “The gene for toxic shock toxinis carried by a family of mobile pathogenicity islands in Staphylococcusaureus” Mol. Microbiol. 29 (2), 527-543): 1 ttatttagca ggaataattagccagattat cgagggagtt ccagggcaatccaaacattg 61 ttatatatgc atttataaaattttcaagat aatttattat tcatacccttgccctttgtt 121 tcaaaattat gccctttttttgcccttgga aacaaccaca ctcctaaattaataggtggt 181 gtggtttgat catttataatataacataaa aacaaccacc cagtaactagtatgagtggc 241 gtagcgacta taacaactctatgttatcaa gatatatgta tatgagtgatgacaaggaag 301 atgtctcctg tgagaccaacagccagatat atggcctctt gccgggctatatagttcact 361 cctactatat acacatgtaattataacata aaaaaataga caagtaccgaagtacctgcc 421 taaataacaa caagattaacatgtgaataa tggaaataaa aagtcagcccgaaggctaac 481 ttacgaatag atgaaaatttgaacacattg ctgtgtctaa aatgattatagcataaataa 541 cgaatatttc cagctcgaaattaatatatt gtaataataa tattttatatctttgttaat 601 aattatttaa ttgatttacataaataataa ttgtaaaatt aatttgtaatcgattgcaaa 661 taagttatag gagaaaataaaatgaataaa aaactattaa caaaaacattgatagcaagt 721 gctttagttt taacaacagtaggttcaggt tttcattctt cttcaaattataatggtatt 781 aataacgttg aaaaagctgagcaaacgaca gataacgcat tgtggaaaaatgtaagagac 841 gctttaaaag acgcgaatattatcgataaa acagataatg aaaatgtcaaggttacgtat 901 aaaatagaaa atggtggagaaaataccata gaaggaacag ttaatttagaaaataftagt 961 acttcaaaca atcctaaaataaaccctcaa aatgttacaa aaattaatataactagaaaa 1021 aatccgaact accctaatattgatgctaat aatacatgga aaaaattaccagaaaaattg 1081 aaagaaaaaa atatagtggaacaacggcga caatgtttca atcttaagtacagaccctaa 1141 agatgagact gtattcggtaaagtaggaga agataaatca aacgtaagcaatagatacat 1201 caatcctaaa gatataaatgaattcaaatc actaaaaata cttttttccgaggcagatta 1261 ctcctgcctc tttctttgaacagtgatatc ttctgatcta tgtaacactcaattacttca 1321 gattctttac ctttaacttcctttaattca tttctctcta tctcctcaaaaagttgtgct 1381 ttttgatttg tgattggagttgggcgtttt ttcatcgcgt tgtttcaattcctttttaag 1441 gtattctaat tctcttctagtcatatcaat tgttttttta cttctcacctttagtgaaat 1501 actcttatcc tttctcttcttgcgttaatg ttgctaatta gtataaaatacatgcgccca 1561 tatattccaa tggtaggacatttaattctg gattttcagc tattttcataaatctattat 1621 ctgataattt gcttaatccaattttcaagc catagcctaa attccccatccactaagtca 1681 ttttgtttca tatggttttaatctacggcc aatctcaaag atagattgaccagcgatgtt 1741 taaagtcata tttcacggatccacatttac gataaacata tctagttacacaatattatc 1801 ccttactgca acacaggacgtttctcagcg taaaaaacac cactagaaagtgactttaaa 1861 gaatataact aattcaaacttatattaatt aatattcttt aaatgaccactcacactttg 1921 ttttttgcta tttgtaactttaaaatgttg tttgaaatct atatttttttgatatagctc 1981 cctatgtaac aaacaatttttaattaatat atatttaaac aagtcaatttagagatcggt 2041 taattcgatt catttaaataatatttatac attctatatg taaacgtttacacatttgaa 2101 gtaaggagaa ttaaaaatga

7. EXAMPLE: EFFECTS OF TOXICAGENTS ON BACTERIAL GROWTH

[0309] In order to demonstrate the methods of the invention, theinventors have expressed and targeted several toxic agents to bacterialpathogens. Toxic agents were selected based on their ability to inhibitthe growth of a pathogen or diseased cell or cause lethality in apathogen or diseased cell. The examples hereinbelow illustrate toxicagents of several naturally occurring phage, plasmid and chromosomallyencoded toxic proteins and demonstrated their effectiveness asantimicrobial therapeutic agents.

[0310] Specifically, several naturally occurring phage, plasmid andchromosomally encoded toxic proteins have been identified and havedemonstrated effective as antimicrobial therapeutic agents, includingbut not limited to SecA, 16S RNA, dicF, sof, dicF antisense, 16Santisense, toxic proteins of the toxin/antidote pairs doc/Phd, gef/Sof,chpBK/ChpB1, or kicB/KicA.

[0311] To illustrate that a toxic agent may be a toxic gene product ofan addiction system toxin, a toxic gene product of achromosomally-encoded toxin, or antisense molecule, nucleic acidsencoding doc, gef, chpBK, kicB or DicF1 were engineered into Transferplasmids for use in the P1 bacteriophage delivery system. Plasmidconstruction was performed by standard methods known in the art.

[0312] As shown in FIG. 3, expression vectors for the cloning of toxicagents were engineered. Specifically, genes encoding the toxic proteinschpBK, kicB, doc and gef under the control of the lacI-regulatedpromoter, were cloned into an E. coli vector containing replicationorigin ColE1 (300-500 copies per cell), pMB1 (15-20 copies per cell) orp15A (10-12 copies per cell) and the selectable marker CAT(chloramphenicol acetyltransferase). However, any selectable markerknown in the art may be used (e.g., bla, ampicillin resistance). Toxicagents were cloned in an E. coli strain that overexpressed the lacIrepressor protein from a lacI expression plasmid. Genes encoding thetoxic proteins chpBK, kicB, doc and gef under the control oflacI-regulated promoter, were obtained by PCR and cloned into an E. colishuttle vector. Lethal agents were cloned in an E. coli strain thatoverexpressed the lacI repressor protein form a lacI expression plasmid.

[0313] Plasmids containing the toxic agents doc or gef were also beenengineered such that a ribosome entry site has been constructed upstreamof the nucleic acids encoding the toxic agent in order to increase thelevels of translation of doc or gef. Plasmids harboring a toxic agentwas called a Transfer plasmid. The Transfer plasmid was constructed suchthat it contained 1) an origin of replication 2) selectable marker 3) P1PAC site, and PAC ABC genes 4) P1 lytic replicon 5) nucleic acidsencoding the toxic agent (e.g., doc, gef, or DicF1).

[0314] Specifically, Transfer plasmids were constructed based onpBluescript (ColE1 origin) and pBBR 122 (broad host range origin) parentvectors. The nucleic acids encoding the toxic agents doc or gef werecloned into the broad host range transfer plasmid. The nucleic acidencoding dicF was cloned into the ColE1 transfer plasmid. The structureof each vector is available. Both doc and gef were placed under lacIregulated promoter. The Transfer plasmids were designed to undergorolling circle replication during the phage lytic cycle.

[0315] A packaging strain (e.g., bacteria cell) was then used to packagethe Transfer plasmid containing the nucleic acid encoding the toxicagent into a bacteriophage phage head. The packaging strain for each ofthe three toxic agents contained the P1 bacteriophage prophage as wellas the Transfer plasmid containing the nucleic acids encoding the toxicagent. In some cases, the packaging strain also contained a thirdplasmid, if necessary, which encoded additional antidote protein whichacted to protect the packaging strain against the toxicity of the toxicagent or the third plasmid encoded additional repressor protein toswitch off the promoter of the Transfer plasmid.

[0316] Thus, the packaging strain (P1 lysogen) was used to package thetransfer plasmid containing the toxic agent (e.g., doc, gef, or DicF1)into phage heads or virions. Phage lysates of the packaging straincontained the infectious bacteriophage virions, and were used to infectbacterial targets in the following manner.

[0317] The P1 lysogen (P1cm C1.100) carrying the transfer plasmid withthe toxic agent (doc or gef or DicF1) was grown at 30° C. in LB, 10 MMMgSO₄, 5 mM CaCl₂, 12.5 μg/ml chloramphenicol until A₄₅₀ reached 0.8 atwhich time the culture was shifted to a 42° C. water bath and aeratedvigorously for 1 h. Chloroform was added and incubation continued for anadditional 20 min at 37° C. The phage stock was clarified bycentrifugation at 4,000 g for 20 min. DNase (1 μg/ml) and RNase (10μg/ml) were added and after incubation at 37° C. 30 min the phage werecentrifuged at 4,000 g 20 min. Phage particles were precipitated fromthe phage stocks by adding NaCl to 1 M and polyethylene glycol 6000 to10% (w/v). After incubation on ice for 2 h the phage were pelleted bycentrifugation at 1,000 g for 15 min. The pellet was carefully dissolvedin 50 mM Tris.Cl pH 7.5, 10 MM MgSO₄, 5 mM CaCl₂, 0.01% Gelatin.Polyethylene glycol was removed by extraction's with chloroform.

[0318] The phage lysates were then used to infect a selected pathogen(e.g., E. coli). Target cells (10⁵ CFU/ml, treated with 10 mM MgSO₄, 5mM CaCl₂) were infected at various M.O.I s (0.1, 1, 10, 100) with eachof the above phage lysate. Following 30 min infection at 30° C. Celldeath was assessed by scoring the plates for the total number of colonyforming units.

[0319] Both types of Transfer plasmids (ColE1 and broad host rangebased) were transferred by the P1 delivery system to various E. colistrains in vitro. The P1 system was also used to deliver the broad hostrange transfer plasmid to P. aeruginosa in vitro. The ColE1 transferplasmid was successfully transferred to E. coli in vivo and the broadhost range transfer plasmid has been delivered in vivo to both P.aeruginosa and E. coli.

[0320] Results indicated that the infection of the bacterial cells withthe phage lysates comprising the infectious virions containing a toxicagent was capable of killing the infected bacterial cells. Further,bacterial cell death was seen to be dose dependent such that higherM.O.I lead to increased cell death. Thus, the methods and compositionsof the invention are useful as antimicrobial agents for treatingpathogenic infections.

[0321] Lethality testing of the toxic agents and has revealed that doc,gef, chpBK and kicB are all bactericidal to E. coli. (See FIG. 4).Specifically, colonies were grown in liquid culture under conditionswhere the expression of the toxic proteins was repressed followingexpression of the protein by induction with IPTG for 1 hour, cultureswere plated out overnight onto agar lacking IPTG. The absence ofcolonies indicates the protein is lethal (see also Table 4 for Results).Constructs were transformed into E. coli and plated onto agar with orwithout 1 mM IPTG. Equal quantities of DNA (500 ng) were alsotransformed into P. aeruginosa, S. aureus and E. faecalis. Mocktransformations were also performed with ‘no DNA.’ +indicates greaterthan 100 colonies, −indicates no detectable colonies. Experiments wererepeated at least two times with the same result. All agents were lethalto E. coli but only doc was toxic in all four. TABLE 4 Assessment of theToxic Proteins in E. coli, P. aeruginosa, S. aureus and E. faecalisusing the Broad Host Range Plasmid. E. coli + P. Construct E. coli IPTGaeruginosa S. aureus E. faecalis Parent vector + + + + + doc + − − − −gef + − + + + chpBK + − − + + kicB + − + + +

[0322] As shown in FIG. 5, the growth of E. coli harboring a docexpression plasmid was demonstrated to be inhibited when the expressionof doc was induced by IPTG. Specifically, cells were grown overnight inLB at 32° C., diluted 1:100 into fresh LB medium and incubated at 32° C.for 180 min. The culture was then divided equally and incubated at 32°in the absence (∘) or presence of 2 mM IPTG (∘) which results in theexpression of the lethal agent doc. Growth was calculated byspectrophotometric measurements with 1 ml samples at OD600.

[0323] Unlike traditional antibiotics which merely slow the growth ofthe bacteria, lethality testing of doc has shown that 99% cell death wasachieved when cells were induced with IPTG for 20 min. A significantreduction (92% cell death) was also demonstrated when the cells wereunder no selective pressure to maintain the doc expression plasmid.Thus, the rapid killing of bacteria reduces the potential for selectivepressure to give rise to resistant strains, which is important ineradicating multidrug resistant bacteria.

Low Resistance to doc

[0324] In order to examine the frequency of resistance to doc, resistantmutants were isolated. The rationale was to select for spontaneousmutations and no mutagens were used. Following prolonged exposure tosublethal concentrations of 40 doc resistant E. coli clones wereisolated. DNA isolated from these clones were tested by transformationto a doc-sensitive cell, however the presence of IPTG did not inducecell killing. This indicates that resistance is due to mutations orrecombination events in the doc expression plasmid, suggesting that achromosomal mutation of the doc target occurs at a very low frequency.

Toxicity to P. Aeruginosa

[0325] Doc and chpBK have been demonstrated to be toxic to P.aerugilosa. Of particular note is that doc had a broad-spectrum activityin both Gram-negative (E. coli and P. aeruginosa) and Gram-positive (S.aureus and E. faecalis) bacteria (see Table 4, above). As seen in Table4, the toxic agent doc killed all species of bacteria tested. Doc, gef,chpBK and kicB were all able to kill E. coli. chpBK killed E. coli andP. aeruginosa.

Development of a Bacteriophage Toxic Agent Delivery System

[0326] A toxic agent delivery system has been achieved for the use of abacteriophage P1 system to package and deliver a Transfer plasmid (SeeFIG. 6A and 6B) to E. coli and P. aeruginosa. FIG. 6A depicts theTransfer plasmid containing the essential signals for packaging (apacsite and a lytic replicon under the control of the P1 P53 promoter), aselectable marker for detection (bla, ampicillin) and ColE1 origin ofreplication in E. Coli. FIG. 6B depicts the lytic replicon whichcomprises the C1 repressor-controlled P53 promoter antisense and geneskilA and repL. The kilA gene contains a 52% in frame deletion. P53antisense is implicated in the stability of the P1 replicon. The methodsof the invention are exemplified herein by two transfer plasmids capableof being efficiently packaged in P1 virions for delivery to pathogenicGram-negative bacteria. Importantly, the delivery system is not underthe constraints of superinfection exclusion (FIG. 7). In order todemonstrate delivery efficiency of the Transfer plasmid by the P1Delivery System to E. coli, the following assay was performed. The E.coli P1Cm clts100 lysogen carrying the transfer plasmid was induced bythermal induction to produce phage particles. Phage lysates were createdwith DNase and RNase and precipitated particles were resuspended in 50mM Tris.Cl pH 7.5, 10 mM MgSO₄, 5 mM CaCl₂, 0.11% gelatin, E. coli C600and E. coli P1 C600 target cells (10⁵ CFU/ml, treated with 10 mM MgSO₄,5mM CaCl₂) were infected with each of the phage lysates. Following 30min incubation at 30° C., infections were plated onto selection platesand antibiotic resistant colonies were scored. Values indicate number ofantibiotic resistant colonies±standard error, n=6.

[0327] Further, the phage-based delivery system is not blocked by aresident phage, such as P1 and lambda, or by compatible plasmids. Thisis important because analyses of environmental samples suggests that upto 40% of P. aeruginosa strains in the natural ecosystems (lake water,sediment, soil and sewage) contain DNA sequences homologous to phagegenomes. The bacteriophage based system is useful to transfer geneticinformation in vivo by delivery of a transfer plasmid expressing anantibiotic marker to E. coli and P. aeruginosa in a mouse peritonitismodel of infection. Plasmid transfer was confirmed by restrictionanalysis and sequencing of the plasmid DNA re-isolated from bacteriarecovered from the intraperitoneal space. Demonstration of transfer invivo has also been obtained.

Develop Bacteriophage P1 Knockouts Able to Package Transfer Plasmid DNAbut Unable to Incorporate P1 DNA

[0328] One consideration of using unodified phage as a delivery vehicleis the potential risk of lysogenic conversion. In order to develop abacteriophage delivery vector which is capable of delivering a Transferplasmid, to a target bacteria, but which is unable to deliver its ownDNA to a target bacteria, a modified P1 phage was developed.

[0329] As shown in FIG. 8, the P1 prophage DNA has been modified togenerate a pac site knockout. The disruption cassette contain anutritional or antibiotic marker flanked by sequences homologous to theP1 prophage the linear fragment was protected from exonuclease attach bythe incorporation of phosphorothioate groups. A double crossover eventbetween the in vitro-altered sequence and the P1 prophage resulted indeletion of the pac site and acquisition of the selectable marker. Thefunction of this knockout serves to inhibit the ability of the P1bacteriophage to package or transfer its own DNA to a target bacteria.

[0330] As shown in FIG. 9, the modified P1 was unable to transfer thechloramphenicol marker associated with its genome, suggesting that phageparticles produced from the pac mutants lack phage DNA. The top panel ofFIG. 9 shows the physical map of the P1 prophage and predicted P1knockout following integration of the disruption cassette at the pacsite. Arrows indicate location of the PCR primers used to verify thereplacement of the P1 pac site with the S. cerevisiae TRP1 gene. Thegels shown the products of the PCRs using P1 specific primers (1, 3, 5and 6) and disruption cassette specific primers (2 and 4) to detecteither the wild type P1 prophage r the P1 knockout. Primers 1 and 3 donot bind within the P1 sequences in the disruption cassette thereforePCR with primer 1+2 and 3+4 only detect a specific integration eventwhich results in replacement of the pac site with the S. cerevisiae TRP1gene.

[0331] As a consequence of the pac site lying within the pacABC operon,the modified phage needed to be complemented in trans with the pacaseenzyme. Construction of the pacABC complementing plasmid is shown in(FIG. 10 and Table 5). TABLE 5 Construction of the pacABC complementingplasmid. c1-pBSK clts100-pBSK Bof-pACYC184 pEDI-clts100-pBSIC1pro-clts100-pBSK clmut-clts100-pBSK Bof-pEDI clts100-pACYC184Bof-C1mut clts100-pACTC184 Bof-C1pro clts100-pACYC184 TpacABCT-Bof-pEDIclts100-pACYC184 TpacABCT-Bof-C1pro clts100-pACYC184 Tpr94pacABCTBof-pEDI clts100-pACYC184

[0332] P1 pacABC were expressed from an early promoter Pr94. Two phageencoded proteins, C1 repressor and Bof modulator, were used to regulateexpression from the Pr94 promoter. Although Bof alone does not bind toDNA, together with C1 it increased the efficiency of therepressor-operator interaction. The c1 repressor has the c1ts 100mutation and was therefore be temperature sensitive. This allowed thecoordinated expression during the phage lytic cycle to the pacABC genes.

[0333] The complementation plasmid allowed P1 pac mutant to package theTransfer plasmid but not its own viral DNA. Complementation with thepacase enzymes did allow the P1 pac mutants to package the transferplasmid, however a portion of the phage particles produced from the pacmutants contained P1 viral DNA. Analysis of the chloramphenicolresistant transductants indicated that the majority were unable toproduce a second round of multiplication, suggesting that they weredefective lysogens. The pac mutants appeared to have acquired apac site,by recombination with the complementing plasmid, thereby enabling themutants to package and deliver its own viral DNA.

[0334] Southern blot analysis verified that the pacABC genes on thecomplementing plasmid had been replaced with the ScTRPI disrupted copy(FIG. 11). Specifically, the P1 mutant lysogens harboring the Transferplasmid and pac ABC complementing plasmid were growth at 32C and diluted1:100 into fresh medium every 16 hours. DAN was extracted on day 1, 2,3, 4, and 5, digested with HindIII and probed with a ScTRP1 EcoR1-BamH1fragment under high stringency conditions.

[0335] In order to prevent reconstruction of functional (pacABC pacenzyme by recombination events between the Transfer plasmid and themodified P1 phage genome, silent mutations were introduced into thecomplementing plasmid as shown in FIG. 12. Silent mutations in thecomplementing plasmid pac site lead to a defective pac site even ifrecombination occurred, and ensured that a defective pac site was beintroduced into the P1 pac knockout (FIG. 12). The 162 bp pac site issufficient to promote pac cleavage and P1 packaging. The positions ofthe hexanucleotide elements with the HEX4 and HEX3 domains are shown byopen boxes the IHF binding site, consensus sequence 5′-AATCAANNANTTA, isindicated. Regulation of pac cleavage involves adenine methylation at5′ - GATC sites. Silent mutations introduced into the pac site areindicated by lower case letters.

In Vivo Delivery of Therapeutic Agents by P1 Virions

[0336] All five animal models listed in Table 6 are exemplified herein.LD50's have been established in the peritonitis models for E. coli andP. aeruginosa, and the doses required are high (10⁷- 10⁸ bacterialcells/animal). Further, a cystic fibrosis model of pseudomonas infectionin mice is used to demonstrate efficacy of the toxic agents and methodsof the invention for treatment of opportunistic lung infectioncharacteristic of this disease. TABLE 6 Animal Models for ProkaryoticGene Therapy Animal Model LD50 E. coli peritonitis 3 × 10⁸ cfuPseudomonas peritonitis 1 × 10⁷ cfu Pseudomonas-burn in mice <10 cfuPseudomonas-burn in rats 1 × 10⁸ cfu Pseudomonas in neutopenic mice 2 ×10³ cfu

Peritonitis Models

[0337] A Transfer plasmid of the invention has been delivered with theP1 delivery system to E. coli and Pseudomonas in vivo in the mouseperitonitis model. Transfer was confirmed by re-isolation of the plasmidfrom bacteria recovered from the intraperitoneal space, and byrestriction analysis of the recovered plasmid. Results demonstrate thatthe delivery vehicles of the invention are capable of delivering thetoxic agents of the invention to a bacterial target without toxicity tothe infected subject.

[0338] The immune response to the phage and phage clearance kinetics invivo has also been examined. Results indicate that single injections of2×10⁹ lysogen forming units (lfu) of P1 phage per mouse resulted in theproduction of anti-phage antibodies in 8-14 days. Two groups of 4 micewere injected intraperitonially (IP) with 2×10⁹ lfu of long circulatingP1 phage. Peripheral blood was sampled by tail clip at 1, 4, 8 and 24hours post injection and titered with E. coli C600 target cells. Thepreviously phage-challenged group had been injected IP with anequivalent dose of the same phage preparation 18 days prior to thisexperiment. The pre-immune group had no prior treatments. This resultedin rapid clearing of the phage in vivo (FIG. 13). However, for humantherapeutic considerations, many infected subjects (especially forpseudomonas) will be immunocompromised and incapable of generatingstrong immune responses. Accordingly, the Therapeutics and compositionsof the invention may be particularly beneficial for such human subjects.

[0339] In addition, a long-circulating variant of P1 was selected bypassage through mice that results in greater than 200 times more phageremaining in circulation at 24 and 30 hours after injection (FIG. 14).Groups of6 mice were injected IP with either 5×10⁸ lfu P1 phage or 5×10⁸lfu long-circulating P1 phage. Peripheral blood was sampled by tail clipat 1, 6, 24, and 30 hours post injection and titered with E. coli C600target cells. The number of viable phage remaining per ml of blood ateach time-point is indicated in FIG. 14. The fold improvement inpersistence in the circulation is given in the last column of the table(lfu long-circulating P1 phage/lfu original P1 phage). Accordingly, useof the long-circulating P1 is within the scope of the invention. Suchvariant may be particularly preferable when increased concentrations ofphage are desired in the circulation of an infected subject. Forexample, it may be desired in the case that the subject has a pathogenor bacterial infection in the blood.

Embryonated Hen Egg Model

[0340] In order to demonstrate efficacy of the toxic agent delivered bythe P1 delivery vehicle, an embryonated hen egg model of infection hasbeen modified from published protocols. Superficially, the hen egg modelof Hartl, A., et al., (1997, “Pseudomonas aeruginosa infection inembryonated hen's eggs” Arzneim.−Forsch. 47(II): 1061-1064), wasmodified in the manner in which the eggs were incubated and the shellsopened and administration was performed. Briefly, eggs were incubated inthe vertical position, wide pole up, with automatic turning in a 90degree arc every 4 h. Shells were opened on the wide pole end, byreinforcing the shell with adhesive tape and cutting a round hole with ascissors through the tape and shell (opening diameter approx. 1 cm). Theunderlying shell membrane was moistened with sterile water, thenpartially removed by tearing off a 1 cm² portion with a sterile forceps,which exposes the transparent chorioallantoic membrane (CAM). The shellwas sealed against moisture loss with more adhesive tape and incubationcontinued for 18-24 h. Viability was assessed at that time by candling(observing the embryo by holding the egg in front of a bright lightsource). Observation of spontaneous movement was evidence of viability.Viable eggs were inoculated by pipetting bacterial suspensions onto theCAM. Therapeutic agents were pipetted onto the CAM or injected throughthe shell at other locations by syringe. Openings in the shell wereresealed with tape, incubation continued, and viability was scored atintervals by candling as above. Bacteria and phage were introduced intothe egg through an opening made in the shell, which was then sealed andgestation continued.

[0341] An embryonated hen egg model was established, as above, whichharbors a variety of advantages as an in vivo system. Specifically, theegg model required very low LD50 (<10 cfu/egg for P. aeruginosa and >50cfu/egg for virulent strains of E. coli), the egg model is also rapid,self contained and provides for an immature immune system. Humanclinical isolates of E. coli and P. aeruginosa (PA01) consistentlyproduce lethal infections in this model at very low doses of bacteria(100-1000 cells) allowing demonstration of the therapeutic agents. Thesetests show efficacy for the toxic agent such as doc in vivo.

[0342] In order to demonstrate the ability of the delivery vehicles ofthe invention to deliver a Transfer plasmid in vivo, a Transfer plasmidcarrying a kanamycin resistance gene was delivered to E. coli and P.aeruginosa in vivo, in mice and in embryonated hen eggs. Resultsindicated that delivery of toxic agent by the P1 system is successful invivo.

[0343]P. aeruginosa Hen Egg Model

[0344] In vivo plasmid transfer in chicken embryos: The Transfer plasmidpBHR was delivered to bacterial cells by P1 phage in vivo in usingembryonated hen eggs, Specifically, groups of six embryonated hen eggswere inoculated via the chorioallantoic membrane on the tenth day ofgestation with the bacteria and phage indicated. P1 lysogen whichharborspDoc, a transfer plasmid which encodes the doc gene. This phagepreparation was a mixture of particles containing either p1 DNA or pDoc.Phage lysates were approximately a ratio of 99:1 P1 containing phageparticles to pDoc containing particles.

[0345] Results demonstrated increased survival of eggs when P1 orP1-pDoc lysates are added immediately after inoculation with humanclinical P. aeruginosa PA01 (FIG. 15).

[0346]E. coli Hen Egg Model:

[0347] A human clinical E. coli isolate which is refractory totransduction with P1 DNA has been found to produce a lethal infection inembryonated hen eggs. This isolate was designated EC-4, and is importantfor two reasons. First, since this strain cannot form a stable P1lysogen, killing of EC-4 cells by doc-carrying phage preparationsdemonstrated the lethal activity of the toxic agent doc.

[0348] Specifically, P1-pDoc lysates killed EC-4 E. coli in vitro moreefficiently than P1-pBHR phage alone (see FIG. 16). Specifically, EC-4cells (500 cfu) were treated with phage containing toxic agent doc (P1-pDoc) or control transfer plasmid pBHR (P1 -pBHR) at the multiplicitiesof infection (MOI) shown in FIG. 16, plated on non-selected media andcounted as a percent of live cells treated with buffer alone. Resultsindicate that the toxic agent doc was able to render E. coli EC-4 lessfit and increase killing of the pathogenic bacteria. Additionally, Ecoli. killing was confirmed in vitro: at a P1 MOI of 500-700, doc wasable to kill the E. coli at a MOI 5-7, i.e. 1% of the total P1 particles(FIG. 16).

[0349] Second, existence of this strain in a random sample of clinicalisolates from local hospital demonstrated that there were pathogenicbacterial strains in the human population which were resistant to lyticphage therapy but susceptible to Toxic agent phage delivery system.Specifically, as shown in Table 7,below three clinical E. coli isolateswere compared for their ability to be transduced with P1 DNA (asindicated by acquisition of chloramphenicol resistance) and transducedwith transfer plasmid DNA (as indicated by acquisition of kanamycinresistance) relative to laboratory E. coli strain C600. All threeclinical isolates were transduced with the Transfer plasmid, but onlytwo became lysogenic with P1. These results indicate that a phageresistant mechanism was preventing transduction of P1 viral DNA wasunable to prevent delivery of the transfer plasmid to EC-4 cells. TABLE7 Clinical Isolates - susceptibility to P1 Transduction. TransductionTransduction with with Transfer E. coli P1 DNA Plasmid DNA C600 (labstrain) 1.9 × 10⁷ lfu 7.7 × 10⁵ lfu C600 in transduced with P1 DNA andthe transfer plasmid EC-1 (Human) 2.0 × 10⁷ lfu 8.5 × 10⁶ lfu EC-1 intransduced with P1 DNA and the transfer plasmid EC-2 (Human)   9 × 10⁶lfu 7.5 × 10⁶ lfu EC-2 in transduced with P1 DNA and the transferplasmid EC-4 (Human) 0 2.8 × 10⁶ lfu EC-4 in transduced with P1 DNA andthe transfer plasmid

[0350] Further, an infection with 2×10³EC-4 cells was cured in eggs by aP1-pDoc lysate treatment given immediately after inoculation with thebacteria at a P1 MOI of 700-800 (doc containing virions were 1% of totalphage particles) (FIG. 17). Specifically, groups of seven embryonatedhen eggs were inoculated via the chorioallantoic membrane on the tenthday of gestation with the bacteria and phage indicated. P1-pDoc phagewas produced from a P1 lysogen which harbors pDoc, a transfer plasmidencodes the doc gene or control transfer plasmid pBHR. This phagepreparation was a mixture of virions containing either P1 DNA or pDoc.Phage lysates were approximately a ratio of 99:1 P1 containing phageparticles to pDoc containing particles. These results indicate that apathogenic infection may be eradicated by a therapeutic of theinvention, such as doc via a P1 delivery vehicle.

Mammalian Animal Models

[0351] Three mouse and rat models are used to demonstrate the efficacyof the Toxic agents of the invention. Each models uses animmunocompromised animal, which is then followed by a bacterialchallenge. The models differ in the route of bacterial challenge and themeans of producing the immune impairment.

[0352] In two models, immune impairment is produced in a bum model.Specifically, a bum of 10-20% total body surface area in humans or otheranimals results in a period of immune impairment, involving nearly allbranches of the immune system, which lasts from 10-14 days. Two burnmodels, well documented in the literature (see, e.g., J. P. Waymack, etal, 1988, “An evaluation of cyclophosphamide as an immunomodulator inmultiple septic animal models” J. Bums and Clinical Rehabilitation9(3):271-274; see also Stieritz, D. D. and Holder, I. A., 1975,“Experimental studies of the pathogenesis of infections due toPseudomonas aeruginosa: Description of a burned mouse model” J. Infect.Dis. 131(6): 688-691) for experimental infections with Pseudomonasaeruginosa, are used to demonstrate the effectiveness of a toxic agenttherapeutic against the types of infections which occur with this typeof wound.

[0353] The third model utilizes the biological modulatorcyclophosphamide to produce an immunocompromised state (leukopenia), inwhich endogenous microflora of the intestinal tract can invade the bodycavity and cause sepsis. This type of sepsis has been documented inhuman patients with immunodeficiency (see, Furuya, et al , 1993,“Mortality rates amongst mice with endogenous septicemia caused byPseudomonas aeruginosa isolates from various sources.” J. MedicalMicrobiology 39: 141-146; Woods, et al, 1997, “Correlation ofPseudomonas aeruginosa virulence factors from clinical and environmentalisolates with pathogenicity in the neutropenic mouse” Can. J. Microbiol.43: 541-551).

[0354] Model 1:Adult Mice, Dorsal Burn, Wound Surface BacterialChallenge

[0355] The first model of use is that of Stieritz, D. D. and Holder, I.A.(1975, J. Infect. Dis. 131(6): 688-691); also see Neely, A. N. andHolder, I. A., 1996, “A murine model with aspects of clinical relevancefor the study of antibiotic-induced endotoxin release in septic hosts.J. Endotoxin Research 3: 229-235.). Young adult female mice, 22-25 g,ICR strain (or possibly Balb/c, CD1, C3HEB/FeJ, C3H/HeJ, C57BL/6, DBA/2,A/J, CBA, C3H/HeN) are anesthetized with pentobarbitol and shaved ofdorsal hair. A heat resistant plastic card with a 1×1.5 inch opening isplaced on the shaved back, 0.5 ml ethanol pipetted onto the exposed skinand ignited for a 10 second burn. The flame is extinguished, and themouse given 1-2 ml saline via intraperitonial (IP) injection as fluidreplacement. This procedure produces a non-lethal, partial thicknessburn covering 12-15% of the body surface area of a 22-25 g mouse (Neelyand Holder, 1996, supra). One hour after the burn, and after mice havereceived analgesia (buprenorphine 2 mg/kg, IM), a small inocula ofbacteria (100 cfu P. aeruginosa) in 0.1 ml saline is injectedsubcutaneously into the wound. Toxic agent treatment agent or placebo isadministered either simultaneously to the same site (also 0.1 ml insaline) or by IP injection (up to 0.5 ml in saline) 1 hour after orshortly before challenge. Animals are observed for sepsis and medicatedfor pain (buprenorphine 2 mg/kg, M) at intervals not exceeding 12 h.Normal diet and water is provided ad libitum. Mortality is expected inuntreated burned groups within approximately 48 h. Blood samples (10-25μl) may be taken at 12-24h intervals by tail bleed to monitor bacterialload. Blood and organs are collected at time of death or euthanasia, tomonitor bacterial load and confirm death from P. aeruginosa sepsis orclearance of infection in treated animals.

[0356] Model 2:Adult Rat, Dorsal Burn, IP or Wound Surface BacterialChallenge

[0357] The second model is that of Waymack et al. (J. P. Waymack, G. D.Warden, J. W. Alexander, P. M. and S. Gonce. , 1988, “An evaluation ofcyclophosphamide as an immunomodulator in multiple septic animalmodels”. J. Burns and Clinical Rehabilitation 9(3):271-274.). Young maleLewis rats (100-125 g) are anesthetized by IP pentobarbitol injection(˜40 mg/kg) and shaved of dorsal hair. The animal is pressed against aheat resistant template that exposes the shaved area (20% of the totalbody surface area). This template is immersed in a 95° C. waterbath for10 seconds. After removal from the waterbath, the animals receive 5-10ml Ringer's Lactate solution by IP injection for fluid maintenancetherapy (approximately one half blood volume is recommended) andbuprenorphine for analgesia (0.1-0.5 mg/kg, every 12 h). This injury isreported to be a full-thickness burn resulting in zero mortality in theabsence of further injury. A 50% lethal dose bacterial challenge (1×10⁸cfu P. aeruginosa in 0.5 ml saline) is introduced by IP injection on day4 post-burn or by painting the bacterial suspension on the wound on day1 post-burn. The IP infection route is reported to produce sepsis within24 h (i.e. day 5 post-burn) with all deaths occurring by day 12post-burn. Therefore, an IP injection demonstration may be terminated onday 12 post-burn. Painting of Pseudomonas on the burn is reported toresult in sepsis 7-8 days after inoculation (day 8-9 post-bum), andsurvival rates are stable by day 20. Euthanasia of animals subjected tothis regimen will be on day 21 post-burn. Normal diet and water will beprovided ad libitum. Some animals are treatment with the therapeutic ofthe invention (P1 phage comprising a Transfer plasmid encoding a toxicagent) which is administered topically to the burn region or by IPinjection. Blood samples (50 100 ul) may be taken by retro-orbital bleedof pentobarbitol anesthetized rats at intervals of 12-24h to monitorbacterial load. Blood and organs are collected at time of death oreuthanasia, also to monitor bacterial load and confirm death from P.aeruginosa sepsis.

[0358] Model 3: Adult Mouse, Antibiotic and Cyclophosphamide Injections,Oral Bacterial Challenge

[0359] This is the model of endogenous septicemia of Furuya etal.(Furuya, N., Hirakata, Y., Tomono, K., Matsumoto, T., Tateda, K.,Kaku, M., and Yamaguchi, K. , 1993, “Mortality rates amongst mice withendogenous septicemia caused by Pseudomonas aeruginosa isolates fromvarious sources.” J. Medical Microbiology 39: 141-146). Mice weighing20-25 g are housed in a sterile environment (e.g., in an isolator) andgiven sterile diet and water. IP injections of sodium ampicillin (200mg/kg) are given on days 1 and 2 to disturb normal intestinal flora andaid colonization by P. aeruginosa. Cyclophosphamide is injected IP (250mg/kg) on days 6 and 9. This dose induces leukopenia without lethalityin the absence of infection. The bacteria are administered to the micein their drinking water on days 2-4. Treatment with therapeutic of theinvention (P1 phage comprising a Transfer plasmid encoding a toxicagent) is started on day 9, and is administered by IP injection. Fecalpellets are be collected before bacterial challenge and at intervalsthroughout the infection to monitor for the absence and presence of P.aeruginosa. The onset of sepsis is expected 2448 h after the second doseof cyclophosphamide (day 11), and approximately 80% mortality isexpected by day 14. Signs of distress in the animals are treated withbuprenorphine (2 mg/kg, twice daily or as needed). Blood samplesobtained by tail bleed may also be taken at 12-24 h intervals after day4. Alternatively, the ampicillin injections can be avoided byintroducing the bacteria by IP injection the day after the finalcyclophosphamide injection (Woods, D. E., Lam, J. S., Paranchych, D. P.,Speert, D. P., Campbell, M., and Godfrey, A. J. , 1997, “Correlation ofPseudomonas aeruginosa virulence factors from clinical and environmentalisolates with pathogenicity in the neutropenic mouse. Can. J. Microbiol.43:541-551). TABLE 8 Therapeutic formulations in the following formatused in the mouse models: Approximate # of Group Challenge Phagemidtreatment survivors  1 Burn or cyclophosphamide (cyc) 1000 × moi agent 16/6  2 Burn or cyc 1000 × moi agent 2 6/6 3-8 Burn or cyc 1000 × moiagent (n) 6/6  9 Burn or cyc +LD₁₀₀ pseudomonas None 0/6 10 Burn or cyc+LD₁₀₀ pseudomonas 1000 × moi agent 1 0/6 6/6 11 Burn or cyc +LD₁₀₀pseudomonas 1000 × moi agent 2 0/6 6/6 12-17 Burn or cyc +LD₁₀₀pseudomonas 1000 × moi agent 3 0/6 6/6

[0360] TABLE 9 A dose response demonstration is performed as follows:Approximate Phagemid # of Group Challenge treatment survivors 1 Burn orcyclophosphamide (cyc) 1000 × moi 6/6 2 Burn or cyc +LD₁₀₀ pseudomonasNone 0/6 3 Burn or cyc +LD₁₀₀ pseudomonas 1000 × moi 0/6 6/6 4 Burn orcyc +LD₁₀₀ pseudomonas  100 × moi 0/6 6/6 5 Burn or cyc +LD₁₀₀pseudomonas  10 × moi 0/6 6/6 6 Burn or cyc +LD₁₀₀ pseudomonas   1 × moi0/6 6/6

[0361] TABLE 10 Ttherapeutic formulations in the following format areused in the rat models: Phagemid Approximate # of Group Challengetreatment survivors 1 Burn 1000 × moi 6/6 2 Burn +LD₁₀₀ pseudomonas None0/6 3 Burn +LD₁₀₀ pseudomonas 1000 × moi 0/6 6/6 4 Burn +LD₁₀₀pseudomonas  100 × moi 0/6 6/6 5 Burn +LD₁₀₀ pseudomonas  10 × moi 0/66/6 6 Burn +LD₁₀₀ pseudomonas   1 × moi 0/6 6/6

[0362] Results of animal demonstrations indicate that the phagetherapeutics comprising a toxic agent of the invention, is suited totreat bacterial infections of a subject. Other animal models known inthe art are within the scope of the invention, including but not limitedto models using calves, pigs, lambs, guinea pig, rabbits, etc. In apreferred aspect of the invention, the subject in need of a therapeuticof the invention is a mammal with a burn injury.

Treatment of Opportunistic Infections in a Murine Model of CysticFibrosis

[0363] The toxic agents of the invention are useful for the treatment ofpathogenic infection such as infections associated with cystic fibrosis.As demonstration of the unility of the invention, a mouse model ofpseudomonas respiratory infection is used which mimics the type ofinfection seen in human cystic fibrosis (CF) patients. This model usesadult (6-8 week old) mice which carry the DF508 mutation in the cftrgene (C57BL/6 DF508 mice ) and their wild type counterparts (C57BL/6mice), or BALB/c adult mice without cftr mutations. The DF508 mutationis one of the most common mutations found in human CF patients, and theC57BL/6 DF508 mice have many symptoms similar to humans with thisdisease. After weaning, DF508 cftr homozygous mutants must be maintainedon a liquid diet of Peptamin (Clintec Nutrition Co., Deerfield, Mich.)and water containing golytely (Braintree Laboratories, Braintree, Mass.)in order to prevent fatal bowel obstructions which are common in thesemice due to their cftr mutation (see Zaidi, T. S., et al, 1999 “Cysticfibrosis transmembrane conductance regulator-mediated corneal epithelialcell ingestion of Pseudomonas aeruginosa is a key component in thepathogenesis of experimental murine keratitis” Infection and Immunity67(3): 1481-1492). BALB/c mice can also be used if C57BL/6 DF508 miceare not available.

[0364] The experimental procedure is as follows (see e.g., Pier, G. B.et al., 1996, “Role of mutant CFTR in hypersusceptibility of cysticfibrosis patients to lung infections” Science 271: 64-67). Adult miceare anesthetized by intraperitoneal injection of a freshly preparedmixture of ketamine hydrochloride (65 mg/kg) and xylazine (13 mg/kg).Then with mice are held in an upright position, and 10 ul of a bacterialsuspension is placed in each nostril (20 ul total). Mice are allowed toregain consciousness and then either observed for survival for up to 72hours, or, sacrificed by CO₂ overdose at various time periods up to 24hours after infection for determination of bacterial loads in varioustissues, especially the lungs. Anesthesia is a necessary part of theinfection procedure. Unanesthetized mice fail to aspirate the inoculumefficiently and do not become infected. Therapeutic phage comprising oneor more toxic agents of the invention are administered, for example,intranasally, intravenously, or intraperitoneally. Mice administered theTherapeutic of the invention survive longer than the untreated controlmice. Accordingly, the toxic agents of the invention may be delivered toa subject harboring a pathogenic (e.g., bacterial) infection for thepurpose of ameliorating or eradicating the infection.

8. EXAMPLE: CONSTRUCTION & CHARACTERIZATION OF SOF SENSE RNA AS A TOXICAGENT

[0365] In order to demonstrate that a toxic agent may be delivered andexpressed using a ribozyme cassette, the inventors have engineered atoxic agent directed against an essential molecule called Sof, anddelivered the toxic agent in a ribozyme cassette to bacterial cells tocause the death of the bacterial cells.

[0366] As described herein above, a toxic agent may be a molecule whichis designed to target an essential molecule of a pathogen or selectedcell. An example of an essential antisense molecule for bacteria is Sof.Sof is an antisense antidote for a chromosomally encoded toxin calledgef Sof normally acts to regulate the levels of gef in the pathogen, andthus allows the cell to survive in the presence of gef The inventors ofthe present invention have designed sense molecules which arecomplementary to Sof The sense molecules against Sof acted to inhibitthe ability of Sof to regulate gef, and thus caused toxicity in thepathogen by allowing the endogenous gef levels to become toxic to thebacteria.

[0367] Specifically, Sof sense was constructed into a triple ribozymecassette (with 5′ and 3′ cis-acting ribozymes). The ribozyme cassettecontaining the Sof sence toxic agent was linked to the LEASHI promoter.The nucleic acids encoding the ribozyme cassette were then used totransform E. coli. Bacterial cells were plated onto LB Amp+IPTG. Plateswere incubated overnight at 37° C. Plates were then scored for thepresence of transformants, size of colonies, growth rate, andmorphological differences.

[0368] Results of these studies indicated that expression of the Sofsense molecules from the ribozyme cassette lead to toxic effects in thetargeted bacteria.

9. EXAMPLE: RIBOZYMES AND RIBOZYME CASSETTES

[0369] The ribozyme cassettes which are particularly usefull in themethods of the invention include but are not limited to the following:

[0370] pClip (the genetic element described in FIG. 19) is amodification of pBluescript, wherein the cassette shown is dropped intothe Not I site in pBluescript. The toxic agent or trans-acting ribozymeis constructed into the Bgl II site (TGCTCT). Liberation of internalribozymes or toxic agents from pClip results in a distribution of thetoxic agent or ribozyme(s) to approximately 20% nuclear and 80%cytoplasmic, when delivered to a eukaryotic cell. pClip is also used totarget prokaryotic cells.

[0371] A second ribozyme cassette/vector that is useful in connectionwith the methods of the invention is pChop. pChop is modified from pClipto convey a more efficient and effective liberation of the internaltrans-acting ribozymes or toxic agents. The pChop ribozyme cassette isdiagramed in FIG. 20. Liberation of internal catalytic core ribozymesfrom pChop increases localization to the nucleus when delivered to aeukaryotic cell.

[0372] A third ribozyme cassette that was useful in connection with themethods of the invention is pSnip. The pSnip multi-ribozyme isconstructed by engineering the pClip cassette 5′ to pChop. In addition,the pSnip multi-ribozyme contains catalytic core sequences with twotrans-acting ribozymes or toxic agents in each cassette. Each pair oftrans-acting ribozymes or toxic agents is linked by a short spacer andstabilized by a hairpin loop located 3′ to the pair. FIG. 21 diagramsthe schematic of the pSnip cassette.

[0373] A trans-acting ribozyme, or antisense toxic agent is synthesizedas reverse complementary overlapping oligodeoxynucleotides, which aredesigned in such a way that when annealed they form single stranded endsidentical to those produced by digestion with the restrictionendonuclease contained with the region between the two cis-actingribozymes. In this particular example the restriction endonucleaserecognition site is that recognized by Bgl II. Essentially any RNA canbe targeted: specificity is conferred by selecting sequences for theribozyme that are reverse and complementary to sequences flanking thechosen cleavage site in the targeted RNA molecule. The toxic agent(s) ortrans-acting ribozymes are then cloned into the cloningregion(polylinker) within the double ribozyme cassette to produce thetargeted toxic agent or ribozyme. Trans-acting ribozymes targeted toprokaryotic sequences have been constructed including, but not limitedto, Escherichia coli: secA (EcosecA, AE000119 U00096), gene X (EcosecA,AE000 U00096)ftsZ(AE000119; U00096), dnaG (AE000388 U00096),rpoA(AE000407 U00096) and tRNA-asp (X14007), Streptomyces lividins secA(Z50195), Enterococcus faecalis, ftsZ (U94707) Pseudomonas putida, dnaG(U385774), Streptomyces coelicolor rpoA (X92107), Staphylococcus warneritRNA-Asp (X66089 S42075), Staphloccocus RNA III.

[0374] The utility of the design using eukaryotic sequences has alsobeen evaluated; a) repetitive B2 transcripts (B2); b) RNA polymerase I(poll); c) Hepatitis B virus (HBV); d) Sonic Hedgehog (SH); e) HumanPapillomavirus E6/E7 protein (HPV); f) RNA polymerase II (pol); g)Insulin-like Growth Factor 1 (IGF1); h) retinoblastoma protein (RB); i)and j) Multicatalytic Proteinase alpha-subunits C3 and C9 (C3 and C9,respectively); k) telomerase (tel); 1) Transforming growth factor beta(TGFβ); m) catalase (CAT); n) Peroxisome proliferation associatedreceptor (PpaRα); and ∘) Cytochrome P₄₅₀ 1E1 (p4501E1); KiSS-1, NudC,Androgen Receptor, and SF-1 transcription factor. Target RNAs (withlocus narnes and accession numbers) as well as the selected target sitesare presented (Table 11). TABLE 11 Summary of Targeted RNAs and TargetSites Functional Target Testing Target RNA EMBL Locus Accession Site invitro in vivo pol II HSRNAP14K Z27113 GTC₈₃ ND ND HBV XXHEPAV X02496GTC₄₃₈ IP + HBV X04615 GTT₁₉₄₄ + + HBV X02496 GTT₁₉₄₆ + + HCV M62321.1GTC₃₂₅ + + RB MUSP105RB M26391 GTC₂₆₄ + + IGF1 HUMIGF1B M37484 GTC₁₈₅ NDND SH MMEVX1 X54239 GTC₅₅₈ IP IP Pol I MUSRPA40 D31966 GTC₄₅₈ + + HPVPPH16 K02718 GTT₁₀₈ IP + C3 RATC3AA J02897 GTT₂₂ + + C9 RNPTSC9 X533304GTC₁₀₁ + + B2 B2-Consensus ## GTT₂₄ + + Tel MMU33831 U33831 CTA₆₃ ND ND

[0375] TABLE 12 Sites Identified on HPV E6/E7 Target RNAs by LibrarySelection GenBank ® Accession Nucleotide Triplet and HPV strains NumberPosition HPV11 M14119 CTC121 HPV16 K02718 TTC110 HPV18 X05015 ATC123HPV11 M14119 ATA443 HPV16 K02718 GTC437 HPV18 X05015 ATC444 HPV11 M14119GTC507 HPV16 K02718 GTC506 HPV18 X05015 ATA507

[0376] TABLE 13 Sites on HBV RNA Identified by Library SelectionNucleotide Triplet and Site GenBank ® Accession Sites on HBV RNA NumberGTC1473 X02496 CTC1534 X02496 CTC1532 X04615, +* ATC1842 X02496 ATC1840X04615, +* GTT1946 X02496 GTT1944 X04615, +* CTC1950 X02496 CTC1948X04615, +# TTC1948 X75663, X75658

[0377] Ribozymes directed to specified target sites in HBV, Pol I andPTEN mRNA were constructed and are listed in Tables X and Y. RegardingTable X, the number following the “Rz-” prefix indicates the cuttingsite in the relevant mRNA. Table Y identifies the flanking sequencesthat correspond to the RNA sequences in the ribozyme that bind to thetarget site. These flanking sequences are found 5′ and 3′ to thecatalytic core, which has the sequence 5′-UUUCGUCCUCACGGACUCAUCAG-′3(SEQ ID NO). The cis elements of the triple ribozymes of the inventionare found in the relevant disclosed vectors. In a preferred embodiment,two ribozymes (the same or different) may be combined with a short (3-15nt) linker between them.

[0378] The catalytic activities of sRz and mRz were determined usingsingle turnover conditions. A trace amount of [³²P]-labeled target-RNAwas incubated with 40 or 200 nM Rz in 5 mM MgCl₂, 20 mM Tris-HCl (pH7.4) at 37 C for 30 minutes, and the cleavage products were separated bydenaturing PAGE (see FIGS. 23 and 24 for in vitro cleavage results forHBV-targeted Rzs). Three of the sRz showed “high” activity during a 30minute cleavage reaction, cleaving between 39-44% of the target RNAusing 40 nM Rz and 48-71% of the target RNA using 200 nM Rz. One of thesRz and one mRz showed “intermediate” activity; they cleaved 8-10% oftarget-RNA at 40 nM Rz or 10-15% at 200 nM. Another mRz was inactive. Inadditional experiments, when the Rz concentration was reduced to 1.6 nM,cleavage products with the 3 highly active sRz were still visible afterPAGE (data not shown). Overall, 92% of sRz showed efficient activitylevels, with 54% being highly active and 38% intermediately active. Incontrast, none of the mRz were highly active; 50% showed intermediate orlow activities, and 50% were inactive (Table X). TABLE X Summary of Rzrelative catalytic activities Activity (high, ###; intermediate, ##;Target Rz, NUH low, #; inactive —) HBV sRz-885, CUC ### sRz-469, CUC ##sRz-408, GUC ### sRz-777, AUC ### m1Rz-247, GUC ## m1Rz-355, GUC Pol IsRz-458, CUA ### sRz-353, AUC ### sRz-595, AUC ## sRz-70, GUC ### PTENsRz-281, AUC ## sRz-681, CUC ## sRz-425, AUC ### sRz-499, GUC ##sRz-774, CUC # m1Rz-127, CUU — m1Rz-151, AUU ## m1Rz-439, UUA —m1Rz-760, AUC — m2Rz-227, AUU ## m2Rz-304, AUC # m2Rz-414, AUA #m2Rz-961, CUA — (###) (##) (#) (—) No. of sRz 7 5 1 % 53.8 38.5 7.7 No.of m1Rz 2 4 % 33.3 66.7 No. of m2Rz 1 2 1 % 25 50 25

[0379] For kinetic analyses, 40 nM Rz and 1 to 100 nM of target RNA wereincubated for various periods (ranging from 20 seconds to 120 minutes),to obtain kinetic data for both single and multiple turnover conditions.Results for the HBV-targeted sRz (FIG. 10 of U.S. ProvisionalApplication No. 60/251,810, incorporated by reference herein) showed aK_(m) of 26 nM, with a K_(cat)/K_(m) of 1×10⁶ (M⁻¹ min⁻¹). Similaranalyses for the other sRz showed K_(cat)/K_(m) values of 0.6×10⁶ (M⁻¹min⁻¹). In comparison, K_(cat)/K_(m) values obtained for mRz to theseand other targets (Benedict et al., 1998, Carcinogenesis 19:1223-1230,Ren et al., 1999, Gene Ther. Mol. Biol. 3:257-269, and Crone et al.,1999, Hepatology 29:1114-1123) are typically an order of magnitudelower.

[0380] To test the effectiveness of the sRz in cells, HepG2 cells (ahuman hepatoblastoma cell line), which can support HBV replication andsecretion after transfection with HBV DNA (they cannot be directlyinfected with virus), were used. HepG2 cells were co-transfected with anHBV DNA construct and HBV-targeted sRz in the CLIP Triple ribozymecassette (Benedict et al., 1998, Carcinogenesis 19:1223-1230, Ren etal., 1999, Gene Ther. Mol. Biol. 3:257-269, and Crone et al., 1999,Hepatology 29:1114-1123). The CLIP cassette encodes 2 cis-acting Rzflanking an internal, transacting Rz targeted to HBV. The 2 cis-actingRz function to release themselves from the primary transcript,liberating the trans-acting internal hammerhead Rz with minimalnon-specific flanking sequences, a process which affords significantadvantages.

[0381] The HBV construct and the Trz constructs were co-transfected intoHepG2 cells, and cultures were analyzed for the effects of sRz on HBVreplication. At 4 and 5 days after transfection with the CLIP constructscontaining sRz777 or sRz885, a dramatic inhibition of secretion of HBVwas observed (FIG. 11, Panel C, of U.S. Provisional Application No.60/251,810, incorporated by reference herein), and this was accompaniedby inhibition of HbsAg secretion (FIG. 11, Panel D, of U.S. ProvisionalApplication No. 60/251,810, incorporated by reference herein) and bymajor reductions in HBV RNA target transcripts (FIG. 11, Panels A and B,respectively, of U.S. Provisional Application No. 60/251,810,incorporated by reference herein). The target sites for sRz777 andsRz885 are located in positions such that all 3 major HBV transcriptsare targeted. For comparison, an mRz408 CLIP construct was alsoemployed, which contained nucleotide substitutions in the 5′ flankingsequence; this Rz showed “intermediate activity” cleaving HBV target atapproximately 20% of the rate at which sRz408 did (not shown), anactivity which was equivalent to that of mRz247. The rnRz408CLIPconstruct was not effective in blocking HBV replication (FIG. 11, PanelsA-D, of U.S. Provisional Application No. 60/251,810, incorporated byreference herein). In addition, a CLIP construct target to anmFold-selected site showed no activity against HBV in this system (datanot shown).

[0382] Two additional repeat experiments with the 777 and 885 CLIPconstructs also demonstrated marked reductions in secretion of HBV (seeFIG. 11, Panel E, of U.S. Provisional Application No. 60/251,810,incorporated by reference herein), although the reductions in HbsAgsecretion and HBV RNA transcripts were more variable. In relatedexperiments, another HBV ribozyme HBV Rz881, which when liberated fromcis elements of the pCHOP vector has the sequence 5′-GGU UCC AGG AUC CAAGAG AGU CUG AUG AGU CCG UGA GGA CGA AAC UCC ACA GUG AAU UCC AAG GGU C-3′(SEQ ID NO) was shown to reduce HBV secretion from, and replication in,from mouse liver cells when complex in liposomes (Templeton et al. 1997)and injected intraperitonealy.

[0383] In summary, the selected Rz targeted to HBV have also been shownto be efficacious in a cell culture model for HBV replication. TABLE YRibozyme Flanking Sequences        9N      6N HBV sRz-4085′-TTCTCGGGG-GCTTGG-3′ sRz-469 5′-GGGCGCACC-TCTTTA-3′ sRz-7775′-TCTGCCTAA-ATCTCT-3′ sRz-885 5′-TGGAGTTAC-TCGTTT-3′ m1Rz-2475′-CGCAGCAGG-TGGAGC-3′ m1Rz-355 5′-CGCGGGACG-CTTTGT-3′ Pol I sRz-705′-TCGCAATGT-CATACT-3′ sRz-353 5′-TCATGCTGA-CCCGTC-3′ sRz-4585′-CCATGCTGC-AAAGAT-3′ sRz-595 5′-ATATCCTCA-GCTCAG-3′ PTEN sRz-2815′-TGAAGACCA-ACCCAC-3′ sRz-425 5′-TTTATTGCA-GGGGCA-3′ sRz-4995′-AAAAGGGAG-ACAATTT-3′ sRz-681 5′-ATATATTCC-CAATTC-3′ sRz-7745′-GTAGAGTTC-CCACA-3′ m1Rz-127 5′-CAGAAAGAC-GAAGGT-3′ m1Rz-1515′-GGAACAATA-GATGAT-3′ m1Rz-439 5′-GCAAATTTT-AAGGCA-3′ m1Rz-7605′-GTGGTGATA-AAAGTA-3′ m2Rz-227 5′-TGAGAGACA-ATAACA-3′ m2Rz-3045′-TAGAACTTA-AAACCC-3′ m2Rz-414 5′-ATTTGTGCA-TTTATT-3′ m2Rz-9615′-TACTCACCC-ACAAAA-3′

Materials & Methods

[0384] In Vitro Cleavage Tests. Rz targeted to the individual sites weretranscribed from double-stranded DNA oligonucleotides using T7 (HBV andPol I) or Sp6 (PTEN) polymerase as described for generation of theguide-RNA library. For standard screening of Rz activity, incubationscontained trace amounts of [³²P]-labeled target RNA, 40 nM Rz RNA, andwere for 30 minutes (or 2 hours) at 37 C in 20 mM Tris-HCl (pH 7.4), 5mM MgCl₂. After incubations, samples were separated in aurea-polyacrylamide gel; the gels were then dried and radioactivity wasanalyzed using a Phosphor-Imager.

[0385] For kinetic analyses, a trace amount of [³²P]-labeled target-RNAwas mixed with unlabeled target-RNA (to yield final concentrations of 1,10 or 100 nM target RNA) and Rz-RNA (40 nM final concentration) andincubations were performed using the same conditions as for the in vitrolibrary selection described above, except that incubation times werevaried (for 20 seconds, 40 seconds, 1 minute, 3 minutes, 10 minutes, 30minutes and 2 hours). The samples were then separated in aurea-polyacrylamide gel, and then dried and analyzed using aPhosphor-Imager.

[0386] Effects of Rz on HBV Replication in Cell Culture. To test theeffectiveness of sRZ in cell culture, HepG2 cells were maintained inminimal essential medium supplemented with 10% heat-inactivated fetalbovine serum, in a humidified incubator at 30 C with 5% CO₂. These cellswere co-transfected with pBB4.5HBV1.3 (a 1.3X unit length HBV DNAplasmid construct; see Delaney & Isom, 1998, Hepatology 28:1134-2246)and either pLSCLIP, pLSCLIPmRz408,pLSCLIPsRz777,or pLSCLIPsRz885(PLSCLIP denotes the CLIP cassette in the LacSwitch vector, fromStratagene). pLSCLIPsRz777 and pLSCLIPsRz885 were constructed byannealing reverse complementary oligonucleotides (CLAW437/CLAW438 andCLAW397/CLAW398, respectively) and then inserting them into the Bgl IIsite of pLSCLIP. pLSCLIPmRz408 was constructed the same way witholigonucleotides CLAW435/CLAW436. However, these oligonucleotides wereinadvertently synthesized so that the 5′ flanking region containedmismatches; subsequent testing in vitro showed that this Rz hadapproximately 20% of the catalytic activity of the sRz408, which wasequivalent to the activity of mRz247, and it was therefore included inthe experiments as an “intermediate” comparison.

[0387] HepG2 cells were transfected using FuGENE6 transfection reagent(Boehringer Mannheim). A total of 5 μg of DNA (0.5 μg pBB4.5HBV1.3 and2.7 μg of the PLSCLIP constructs), 24 μl of enhancer, and 30 μl ofEffectene transfection reagent. The cells were incubated in theDNA/reagent mixture in serum-containing medium for 6 hours.

[0388] For Northern blot analyses, total RNA was isolated fromtransfected HepG2 cells four and five days post-transfection(Chomczynski & Sacchi, 1987, Anal Biochem 162:156-159), and NorthernBlot analysis was performed using 10 μg of total RNA as described (Daviset al., 1986, Preparation and analysis of RNA from eukaryotic cells. In:Basic Methods in Molecular Biology, New York: Elsevier SciencePublishing Co., Inc., 129-156). Hybridization was performed using a[³²P]-radiolabeled HBV probe generated by random priming (withBoehringer Mannheim Random Prime DNA Labeling ktis). The blots wereprobed simultaneously for HBV and GAPDH transcripts. Followinghybridizations, the blots were rinsed under high-stringency conditionsand exposed for audoradiography.

[0389] For analysis of secreted extracellular HBV DNA, medium wascollected on day 4 and day 5 post-transfection, and centrifuged at6,000×g for 5 minutes to remove cellular debirs. Triplicate samples werepooled and HBV particles were precipitated and analyzed as described(Wei et al., 1996 J. Virol. 70:6455-6458). Viral pellets wereresuspended in PBS and digested with Proteinase K, then extracted withphenol/chloroform. DNA was precipitated with 0.1 volume of 3 M sodiumacetate and 1 volume of isopropanol. Ten micrograms of tRNA was added asa carrier during precipitation. Pellets were resuspended in TE anddigested with 0.5 mg/ml RNase for 1 hour. DNA was then analyzed byelectrophoresis and Southern blotting, followed by autoradiography.

[0390] For analysis of secreted HBV Surface Antigen (HbsAg), detectionwas performed by radioimmunoassay using a Sorin Diagnostics kit. Mediumfrom transfected cells was collected and centrifuged at 6,000×g toremove cellular debris. Total counts were compared for analysis.

10. EXAMPLE: PREFERRED TARGET SEQUENCES AND SEVERAL PREFERRED DNAZYMES

[0391] The human papilloma virus (HPV) and hepatitis B virus (HBV)target sites identified in the instant disclosure are useful as sitesfor the hybridization of antisense oligonucleotides, DNAzymes orribozymes targeted against these sites. Preferred targets are HPV E6/E7mRNA or the core, pre-core or polymerase-encoding sequences of hepatitisviruses.

[0392] A preferred embodiment of a DNAzyme specific for HPVl 6, denotedHPV16-Dz57 is the following: 5′-TGTGGTAAGGCTAGCTACAACGATTTCTGGG-3′. Thecatalytic core is underlined and the flanking 5′ and 3′ sequenceshybridize under physiological conditions to the target sequenceidentified as SEQ ID NO:AA.

[0393] In general, the DNAzymes of the present invention are made byadding 5′ and 3′ flanking sequences to a catalytic core sequence. The 5′and 3′ flanking sequences are designed so as to be complementary to atarget sequence of interest. For example, the target sequencesidentified in Table 14 (SEQ ID NOs:AA-BD) maybe used for the design ofDNAzymes specific for each of the disclosed target sites. Table 14 showsDNA sequences corresponding to the sense orientation of target mRNAs.The DNAzymes in Table 15 have flanking sequences that are the reversecomplement of the Table 14 sequences. Underlined in each target sequence(SEQ ID NOs:AA-BD) is the cleavage site where the DNAzyme cuts the RNAcorresponding to the target site. A DNAzyme of the present invention isdesigned by identifying the nucleic acids flanking but not including theunderlined cleavage site of interest in a specific target sequence anddesigning complementary nucleic acid sequences that will bind to thetarget sequence flanking the cleavage site. The complementary sequencesmay be synthesized chemically using techniques well known in the art andjoined to the 5′ and 3′ ends of the catalytic core of the DNAzyme. Thecomplementary nucleic acid sequences that are attached to the 5′ and 3′ends of the catalytic core of the DNAzyme provide specificity for thetarget site of interest because the 5′ and 3′ flanking sequencesspecifically hybridize under physiological conditions to the target siteof interest. The 5′ and 3′ flanking sequences that are attached to theDNAzyme catalytic core may be from 6-15 nucleotides in length, morepreferably, they may be from 7-10 nucleotides in length, even morepreferably, they may be from 8-9 nucleotides in length. In specificembodiments of the instant invention, the DNAzymes set forth in Table 15(SEQ ID NOs:BE-BQ) are designed to be specific for the HPV targetsequences disclosed as SEQ ID NOs:AA-AM, respectively. As will bereadily apparent to a person of ordinary skill in the art, the design ofDNAzymes specific for the target sequences of the instant specificationmay be accomplished using the strategy described in detail above. Thespecific exemplifications herein are in no way intended to limit thescope of the invention. TABLE 14 Target Sequences Accession Cutting SEQTarget No. Site Target Sequence ID NO HPV16 K02718 136 CCCAGAAAGTTACCACAAA 145 TTACCACAGTTATGCAC AB 227 GACGTGAGGTATATGAC AC 264CATAGTATATAGAGATG AD 328 ATTAGTGAGTATAGACA AE 352 TATAGTTTGTATGGAAC AF492 TATAAGGGGTCGGTGGA AG 504 GTGGACCGGTCGATGTA AH 3915 GTGCTTTTGTGTGTCTGAI 4015 GCCTCTGCGTTTAGGTG AJ HPV11 M14119 128 GCCTCCACGTCTGCAAC AK 436GCCGTTGTGTGAAATAG AL 505 GTGGAAGGGTCGTTGCT AM HBV V01460 1473TTCTCGGGGTCGCTTGG AN 1842 TCTGCCTAATCATCTCT AO 1950 TGGAGTTACTCTCGTTT APHPV16 K02718 110 TGCAATGTTTCAGGACC AQ 437 GCCACTGTGTCCTGAAG AR 506GTGGACCGGTCGATGTA AS HPV11 M14119 121 AAAGATGCCTCCACGTC AT 443TGTGTGAAATAGAAAAA AU 507 GTGGAAGGGTCGTTGCT AV HPV18 X05015 123CTTTGAGGATCCAACAC AW 444 ACCGTTGAATCCAGCAG AX 507 TGGGCACTATAGAGGCC AYHPV16 K02718 138 CCCAGAAAGTTACCACA AZ 147 TTACCACAGTTATGCAC BA 266CATAGTATATAGAGATG BB 330 ATTAGTGAGTATAGACA BC 354 TATAGTTTGTATGGAAC BD

[0394] TABLE 15 DNAzymes specific for HPV target sequences. The boldunderlined portion of each sequence represents the catalytic core of theDNAzyme; the 5′ and 3′ flanking sequences hybridize to the targetsequence. SEQ Target ID No. DNAzyme NO HPV16-1 5′-TGTGGTAAGGCTAGCTACAACGA TTTCTGGG-3′ BE HPV16-2 5′-GTGCATAA GGCTAGCTACAACGATGTGGTAA-3′ BF HPV16-3 5′-GTCATATA GGCTAGCTACAACGA CTCACGTC-3′ BGHPV16-4 5′-CATCTCTA GGCTAGCTACAACGA ATACTATG-3′ BH HPV16-5 5′-TGTCTATAGGCTAGCTACAACGA TCACTAAT-3′ BI HPV16-6 5′-GTTCCATA GGCTAGCTACAACGAAAACTATA-3′ BJ HPV16-7 5′-TCCACCGA GGCTAGCTACAACGA CCCTTATA-3′ BKHPV16-8 5′-TACATCGA GGCTAGCTACAACGA CGGTCCAC-3′ BL HPV16-9 5′-CAGACACAGGCTAGCTACAACGA AAAAGCAC-3′ BM HPV16-10 5′-CACCTAAA GGCTAGCTACAACGAGCAGAGGC-3′ BN HPV11-1 5′-GTTGCAGA GGCTAGCTACAACGA GTGGAGGC-3′ BOHPV11-2 5′-CTATTTCA GGCTAGCTACAACGA ACAACGGC-3′ BP HPV11-3 5′-AGCAACGAGGCTAGCTACAACGA CCTTCCAC-3′ BQ

11. EXAMPLE: EFFECTS OF ANTISENSE OLIGONUCLEOTIDES TARGETED TO AN HBVSITE IN TRANSGENIC MICE

[0395] As shown in the data presented in the following Tables 16 to 21,a DNAzyme (Dz879, 5′-GAG AGT AAG GCT AGC TAC AAC GAT CCA CAG T-3′, SEQID NO:) or its catalytically inactive counterpart (i.e., an antisenseoligonucleotide, rnDz879 5′-GAG AGT AAG CCT AGC TAC TAC GAT CCA CAGT-3′), was effective in reducing HBV replication and secretion in atransgenic mouse that expresses human HBV, i.e., in vivo. TABLE 16Effects of DNAzyme (Dz879) or the catalytically inactive counterpart(m879) on serum HBV genome equivalents in HBV Transgenic Mice. 50 ug ofDz879 or m879 were administered in asialofetuin-coated liposomes twiceper week for 2 weeks, and sacrificed 48 h after the final treatment. Asis evident, both Dz879 and m879 were effective in reducing HBV secretionin vivo after 2 weeks of treatment. However, the effect was diminishedafter 5 weeks, presumably because of an immune response to theasialofetuin. Serum HBV Genome Equivalents/ml (×10⁻³) Group 1 Group 3Group 5 Group 7 Group 9 Dz, 2 weeks Dz. 5 weeks mDz, 2 weeks mDz, 5Control week 0.4 ± .38 3.29 ± 3.35 0.74 ± 0.85 4.10 ± 3.19 6.57 ± 3.31 Pvalues, Student's t-test 0.0025 0.072 0.003 0.180

[0396] TABLE 17 Effects of Dz879 and m879 on HBV Core Ag in liver of HBVTransgenic Mice. Dz879 and m879 were administered in asialofetuin-coatedliposomes as described. Liver tissue was obtained, fixed, processed, andimmunohistochemistry was performed for HBV Core antigen around centralveins. As is evident, there is a dramatic reduction in staining for HBVCore antigen after 2 weeks. In addition, the intensity of staining wasalso greatly reduced, indicating an even more marked effect than isshown by the cytoplasmic staining numbers. Animals: Female Transgenicmice (founder 1.3.32) Treatment schedule: twice per week, (Tue, Friday)× 2 or 5 weeks Virus: Human hepatitis B virus Treatment route: i.p.Drug: Prepared at Penn State sent to USU Experiment duration: 2 or 5weeks Mean HbcAg-stained cytoplasms/total cells^(a) ± standard devation(n^(b)) Treatment Day 14^(c) Day 35 CL-ASF-DNAzyme 0.04 ± 0.05 (10)***0.14 ± 0.12 (10)*** CL-ASF-DNAzyme 0.15 ± 0.13 (10)*** 0.30 ± 0.15 (9)*mutant No Treatment 0.47 ± 0.18 (10)

[0397] TABLE 18 Effects of Dz879 and m879 on HBV RNA Transcripts in HBVTransgenic Mice. Dz879 and m879 were administered as described, andliver tissue was extracted for RNA. RNA was analyzed by Northern blotanalysis followed by densitometry; results showed major reductions inHBV RNA transcript levels (all transcripts, as was the case with cellculture results). Animals: Female Transgenic mice (founder 1.3.32)Treatment schedule: twice per week, (Tue, Friday) 2 weeks Virus: Humanhepatitis B virus Treatment route: i.p. Drug: Prepared at Penn Statesent to USU Experiment duration: 2 or 5 weeks Treatment Relative LiverHBV RNA^(a) ± SD (n^(b)) CL-ASF-DNAzyme  7.0 ± 3.5 (10)* CL-ASF-DNAzyme 9.4 ± 5.7 (10)* mutant No Treatment 16.0 ± 5.6 (10)

[0398] TABLE 19 Effects of Dz879 and m879 on HBV liver DNA in TransgenicMice. Dz879 and m879 were administered in asialofetuin-coated liposomes.Liver tissue was extracted for DNA, and HBV genomic DNA was quantitatedby cross-over PCR. Administration of Dz879 and m879 resulted in adramatic reduction in HBV liver DNA. Animals: Female Transgenic mice(founder 1.3.32) Treatment schedule: twice per week, (Tue, Friday) X 2or 5 weeks Virus: Human hepatitis B virus Treatment route: i.p. Drug:Prepared at Penn State sent to USU Experiment duration: 2 or 5 weeksLiver HBV DNA Mean log₁₀ fg/ug cel DNA ± sd (n^(a)) Treatment Day 14^(b)Day 35 CL-ASF-DNAzyme  1.9 ± 0.22 (10)* NT^(c) CL-ASF-DNAzyme 1.75 ±0.21 (10)* NT^(d) mutant No Treatment 4.11 ± 0.34 (8)

[0399] TABLE 20 Effects of Dz879 or a control DNAzyme on HBV liver DNAin Transgenic Mice. This represents a repeat experiment showing markedeffects of the DNAzyme on liver HBV DNA. Further experiments are beingperformed to determine if the catalytic core of the DNAzyme, or itsrapid breakdown from oligonucleotides to monophosphate nucleotides maybe responsible for the observed effects in the placebo group. GeneExpression analysis has shown increased expression of deoxycytidinekinase, for example, while indicating that cytokines are not responsiblefor the effects. Animals: Female Transgenic mice (founder 1.3.32)Treatment schedule: see below Virus: Human hepatitis B virus Treatmentroute: i.p. Drug: Prepared at Penn State sent to USU Experimentduration: 3 weeks Liver HBV DNA mean log₁₀ fg/ug cell Therapeutic DosageTreatment Schedule DNA ± SD (n^(a)) Group 13, DNAzyme 50 ug/ Treat day0, 2, 6, 9, 1.87 ± .059*** injection necropsy day 12 Group 15, CL-ASF-Treat day 0, 2, 6, 9, 1.79 ± 0.21*** placebo necropsy day 12 Group 17,untreated — 4.11 ± 0.34

[0400] TABLE 21 Effects of Dz879 or a control DNAzyme on HBV CoreAntigen Staining in Transgenic Mice. Animals: Female Transgenic mice(founder 1.3.32) Treatment schedule: see below Virus: Human hepatitis Bvirus Treatment route: i.p. Drug: Prepared at Penn State sent toExperiment duration: 3 weeks Mean HbcAg- stained^(a ±) standardTherapeutic Treatment deviation Cytoplasm/Total cells Dosage Schedule(n^(b)) Group 13, DNAzyme 50 ug/ Treat day 0, 2, 6, 9, 0.08 ± 0.09 (7)*injec- necropsy day 12 tion Group 15, CL-ASF- Treat day 0, 2, 6, 9, 0.09± 0.09 (8)* placebo necropsy day 12 Group 17, untreated — 0.34 ± 0.25(6)

12. EXAMPLE: USE OF MULTIPLE DNAZYMES TO INHIBIT PAPILLOMA GROWTH INCOTTONTAIL RABBITS

[0401] DNAzymes were also tested in the model cottontail rabbit system.Sections of cottontail rabbit skin were lightly abraded, and asuspension of Shope Papilloma Virus was applied. After 10 days, DNAzymesin saline solution were applied topically at 30 μg/day for 4 weeks, andthen at 60 μg/day for 2 additional weeks. The following DNAzymes weretested individual and as mixture: Group L1 (SEQ ID NO WW); Group L2 (SEQID NO XX); Group L3 (SEQ ID NO YY). The target sites for the DNAzymeswere selected by homology to previously-identified HPV sites. Acatalytically-defective DNAzyme (Group mL2, SEQ ID NO ZZ) was alsotested. Papillomas were then measured, and mean volumes calculated. Asshown in FIG. 25, the individual DNAzymes and the catalyticallydefective DNAzyme were not effective in inhibiting papilloma growth.However, a mixture of all three DNAzymes (L1/L2/L3) was highly effectivein reducing papilloma growth.

[0402] TGCCGGGAGGCTAGCTACAACGATCGGGGCT (SEQ ID NO WW) is LS 1 DNAzyme toShope Papilloma Virus (GenBank® Accession No. AJ404003) with cleavagesite at nucleotide 614.

[0403] CACAGAAAGGCTAGCTACAACGAAGACTGAA (SEQ ID NO XX) is LS2 DNAzyme toShope Papilloma Virus (GenBank® Accession No. AJ404003) with cleavagesite at nucleotide 935.

[0404] TATAGAAGGGCTAGCTACAACGAAGCCCTGC (SEQ ID NO YY) is LS3, a DNAzymeto Shope Papilloma Virus (GenBank® Accession No. AJ404003) with cleavagesite at nucleotide 1006.

[0405] CACAGAAAGCCTAGCTACTACGAAGACTGAA (SEQ ID NO ZZ) is mLS2, acatalytically defective DNAzyme to Shope Papilloma Virus (GenBank®Accession No. AJ404003).

13. EXAMPLE: DELIVERY AND IN VIVO TESTING

[0406] Biologic Delivery

[0407] The toxic agents and/or ribozymes of the present invention may bedelivered by a wide variety of viral vectors and bacteriophage asdescribed herein, and exemplified herein above.

[0408] In one embodiment of the invention, a toxic agent is encoded in aTransfer plasmid, and is used in connection with a P1 bacteriophagedelivery system. Such Transfer plasmid preferably contains 1) an originor replication 2) selectable marker 3) P1 PAC site and PAC ABC genes 4)P1 lytic replicon 5) nucleic acids encoding one or more toxic agents ofthe invention. In a preferred embodiment of the invention, thebacteriophage P1 prophage (P1 plasmid) is engineered such that viral DNAcan not be packaged into virions, such as, for example, by deletion ofthe PAC site from the P1 plasmid.

[0409] In another embodiment, the toxic agents and/or ribozymes may bedelivered via a plasmid encoding the toxic agents and/or ribozymes, aplasmid origin of replication, a selectable marker for plasmidmaintenance, the minimal lambda origin of replication, and cos sites,which are required for packaging of DNA into lambda virions. Thisplasmid is maintained in a lambda lysogen that is defective inintegration/excision and recombination functions. The defective lysogenprovides all of the replication factors needed to activate the lambdaorigin of replication on the plasmid and all of the structuralcomponents needed to form mature virions; however, the lysogen is notable to replicate and package its own DNA into the virions. The lysogenalso carries the cI⁸⁵⁷ temperature-sensitive repressor mutation.Induction of the lysogen by temperature shift to 42° C. or by othermeans, such as exposure to 5J/m2 of ultraviolet radiation will mobilizethe plasmid and result in its replication and packaging into lambdavirions. The virions can then be harvested, purified free of E. coliproteins and be used to deliver the toxic agents and/or ribozyme gene(s)to E. coli. Similar methods are performed for Pseudomonas aeruginosa inorder to deliver a toxic agent and/or ribozyme to P. aeruginosa.

[0410] Abiologic Delivery

[0411] Abiologic delivery of the toxic agent and/or ribozymes isaccomplished with constructs that have been engineered to be expressedwithin the targeted tissue or pathogen. Briefly, the genetic elementcontaining the promoter and the toxic agent and/or ribozyme(s) arecomplexed with cationic liposomes (Lipofectamine--Gibco BRL) in a 1:10ratio and are introduced into test animals by either single or multipleinjection of 0.2 ml total volume nucleic acid-liposome mixture.

[0412] Prophylactic Administration and Prevention of Acute BacterialInfection

[0413] Following the demonstration that toxic agents and/or ribozymes ofthe present invention have an in vitro biological activity (eitherdirectly on bacterial cultures or in an infectious tissue culture cellassay system), the effectiveness of the toxic agents and/or ribozymes,is shown in in vivo model systems, e.g., as described above. Todemonstrate the efficacy of toxic agents and/or ribozymes of theinvention in vivo, experimental animal model systems are utilized, suchas those described herein. For an initial demonstration of the efficacyof the toxic agents and/or ribozymes in vivo, mice are infected with amicrobial pathogen which has previously been shown to be sensitive tothe toxic agents and/or ribozymes construct(s) and the effect of toxicagents and/or ribozymes administered in vivo is determined. In the firstseries of in vivo trials, one determines the effectiveness of toxicagents and/or ribozymes at preventing an acute infection in a murinemodel system when the toxic agents and/or ribozymes is added directly tothe microbe prior to administration in vivo.

[0414] The next series of trials demonstrates that the administration oftoxic agents and/or ribozymes after infection is effective at preventingan acute bacterial infections. In addition to the clinical status ofinfected mice, tissues obtained at necropsy are examined histologicallyand the presence of replicating microorganism in tissue samples isdetermined by standard methodology. Animals can be infected by variousroutes (systemic and/or mucosal) and the toxic agents and/or ribozymesare delivered over time after infection by systemic, mucosal, or topicalroutes. Both abiologic as well as biological delivery of the toxicagents and/or ribozymes is used. The demonstration of a positive effectof the toxic agents and/or ribozymes in controlled experimental modelsystem provides compelling evidence for the efficacy of the preparationand determines whether or not the preparation warrants evaluation underconditions of standard clinical trials.

[0415] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

[0416] Throughout this application various publications are referenced.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

1 92 1 66 DNA Artificial Sequence LEASH1 promoter 1 gatcctcagaaaattatttt aaatttccaa ttgacattgt gagcggataa caatataatg 60 tgtgga 66 2 74DNA Artificial Sequence modified rrnB promoter 2 agaaagcaaa aataaatgcttgacactgta gcgggaaggc gtataatgga attgtgagcg 60 gataacaatt caca 74 3 492DNA Artificial Sequence ANR promoter 3 actcgcggat catcttcacc atcggccgcaactcctgcgg gatatcctcg tcctcctcct 60 ccaccggcac ccccatggta gcggccagctcgcgccctgc ctgggaaagc tgtacatgct 120 gatcggcggc gtcggtgccg gcggccgggtcttccgcctg ctcggcggtg ccggtccgtg 180 cggccttggc gtccgcggcg gcgcgcgatgagggcggcac ctgggtggtg atccagccac 240 tgagggtcaa cattccagtc actccgggaaaaatggaatt cttccattgg atcggcccac 300 gcgtcgcgaa cttgagcccc cttttcgtcgccccttgaca gggtgcgaca ggtagtcgca 360 gttgtttgac gcaagtcact gattggaaacgccatcggcc tgtcagaaat ggtcgttgcc 420 agacctatgg ctggcacccg catcgcggctgcgttaccct tactcctgtt gtgcctttaa 480 cctagcaagg ac 492 4 1113 DNAArtificial Sequence ProC promoter 4 aattcctcga agtccttgcg ctgcttgtcgttcatgatgt cgtagatcag cgcatgcacc 60 tgcttgtgtt ccagcggtgg caggttgatccggcgtacat cgccatccac ccggatcatg 120 ggtggcaggc cggcggagag gtgcaggtccgaagcgccct gtttggcact gaaggcgagc 180 agctcggtaa tatccatggg actccccaattacaagcaag caggtagaat gccgccaaag 240 ccgccgtctc ggacaaggaa aacaccggatgagccagggt gcttccagga cacgcgtggt 300 gtcctgcgcc agacgcggaa cctcgacactggaacaggaa gatggccatc gaggccggcg 360 gtttcgaggg cgtcgagccg acgccgaccgcacttccata gggcgcaggt aatgtccacg 420 atagcagaga atattgcaaa ggttgccgcgcgcatccgtg aggcagcgca agctgcgggg 480 cgcgatccgg ccacggtcgg cctgctcgccgtgagcaaga ccaagcccgc cgccgcggtg 540 cgcgaggcgc acgccgccgg ccttcgcgacttcggcgaaa actacctgca ggaggccctc 600 ggcaagcagg ccgaactggc cgacctgcccttgaactggc acttcatcgg ccccatccag 660 tcgaacaaga cgcggcccat cgccgagcatttccagtggg tgcactcggt ggaccggttg 720 aagatcgcgc agcgcctgtc ggagcaacgcccggccgggc tgccgcccct gaatgtctgc 780 ctgcaggtca acgtcagcgg cgaagccagcaagtccggct gcgcccccga ggacctgccg 840 gccctggccg aggccgtgaa gcaactgcccaacctccgat tgcgtggcct gatggccatc 900 cccgaaccca ccgccgaacg cgccgcgcaacacgccgcgt tcgcccgcct gcgcgaactg 960 ctgctggacc tgaaccttgg cctggacaccctgtccatgg gcatgagcga cgacctcgag 1020 gcagccatcg gcgaaggtgc gacctgggtccgcatcggta ccgccctgtt cggcgcccgc 1080 gactacggcg cgccggcttc ttgaatgaatccc 1113 5 66 DNA Artificial Sequence ARC promoter 5 ctagagctattgatgtggat caacattgtc cactagccgc tgccgcctaa tctccagaat 60 tgtgag 66 62120 DNA Staphylococcus aureus 6 ttatttagca ggaataatta gccagattatcgagggagtt ccagggcaat ccaaacattg 60 ttatatatgc atttataaaa ttttcaagataatttattat tcataccctt gccctttgtt 120 tcaaaattat gccctttttt tgcccttggaaacaaccaca ctcctaaatt aataggtggt 180 gtggtttgat catttataat ataacataaaaacaaccacc cagtaactag tatgagtggc 240 gtagcgacta taacaactct atgttatcaagatatatgta tatgagtgat gacaaggaag 300 atgtctcctg tgagaccaac agccagatatatggcctctt gccgggctat atagttcact 360 cctactatat acacatgtaa ttataacataaaaaaataga caagtaccga agtacctgcc 420 taaataacaa caagattaac atgtgaataatggaaataaa aagtcagccc gaaggctaac 480 ttacgaatag atgaaaattt gaacacattgctgtgtctaa aatgattata gcataaataa 540 cgaatatttc cagctcgaaa ttaatatattgtaataataa tattttatat ctttgttaat 600 aattatttaa ttgatttaca taaataataattgtaaaatt aatttgtaat cgattgcaaa 660 taagttatag gagaaaataa aatgaataaaaaactattaa caaaaacatt gatagcaagt 720 gctttagttt taacaacagt aggttcaggttttcattctt cttcaaatta taatggtatt 780 aataacgttg aaaaagctga gcaaacgacagataacgcat tgtggaaaaa tgtaagagac 840 gctttaaaag acgcgaatat tatcgataaaacagataatg aaaatgtcaa ggttacgtat 900 aaaatagaaa atggtggaga aaataccatagaaggaacag ttaatttaga aaatattagt 960 acttcaaaca atcctaaaat aaaccctcaaaatgttacaa aaattaatat aactagaaaa 1020 aatccgaact accctaatat tgatgctaataatacatgga aaaaattacc agaaaaattg 1080 aaagaaaaaa atatagtgga acaacggcgacaatgtttca atcttaagta cagaccctaa 1140 agatgagact gtattcggta aagtaggagaagataaatca aacgtaagca atagatacat 1200 caatcctaaa gatataaatg aattcaaatcactaaaaata cttttttccg aggcagatta 1260 ctcctgcctc tttctttgaa cagtgatatcttctgatcta tgtaacactc aattacttca 1320 gattctttac ctttaacttc ctttaattcatttctctcta tctcctcaaa aagttgtgct 1380 ttttgatttg tgattggagt tgggcgttttttcatcgcgt tgtttcaatt cctttttaag 1440 gtattctaat tctcttctag tcatatcaattgttttttta cttctcacct ttagtgaaat 1500 actcttatcc tttctcttct tgcgttaatgttgctaatta gtataaaata catgcgccca 1560 tatattccaa tggtaggaca tttaattctggattttcagc tattttcata aatctattat 1620 ctgataattt gcttaatcca attttcaagccatagcctaa attccccatc cactaagtca 1680 ttttgtttca tatggtttta atctacggccaatctcaaag atagattgac cagcgatgtt 1740 taaagtcata tttcacggat ccacatttacgataaacata tctagttaca caatattatc 1800 ccttactgca acacaggacg tttctcagcgtaaaaaacac cactagaaag tgactttaaa 1860 gaatataact aattcaaact tatattaattaatattcttt aaatgaccac tcacactttg 1920 ttttttgcta tttgtaactt taaaatgttgtttgaaatct atattttttt gatatagctc 1980 cctatgtaac aaacaatttt taattaatatatatttaaac aagtcaattt agagatcggt 2040 taattcgatt catttaaata atatttatacattctatatg taaacgttta cacatttgaa 2100 gtaaggagaa ttaaaaatga 2120 7 177DNA Artificial Sequence P1 pac site 7 ccactaaaaa gcatgatcat tgatcactctaatgatcaac atgcaggtga tcacattgcg 60 gctgaaatag cggaaaaaca aagagttaatgccgttgtca gtgccgcagt cgagaatgcg 120 aagcgccaaa ataagcgcat aaatgatcgttcagatgatc atgacgtgat cacccgc 177 8 45 DNA Artificial Sequence DicF1molecule 8 caggcgacag gtatagtttc tctccgattt gtgcctgtcg cctgc 45 9 14 DNAArtificial Sequence consensus sequence 9 ggaggtgnnn natg 14 10 15 DNAArtificial Sequence DNAzyme 10 ggctagctac aacga 15 11 23 RNA ArtificialSequence catalytic core 11 uuucguccuc acggacucau cag 23 12 67 RNAArtificial Sequence HBV Rz881 12 gguuccagga uccaagagag ucugaugaguccgugaggac gaaacuccac agugaauucc 60 aaggguc 67 13 15 DNA ArtificialSequence HBV sRz-408 13 ttctcggggg cttgg 15 14 15 DNA ArtificialSequence HBV sRz-469 14 gggcgcacct cttta 15 15 15 DNA ArtificialSequence HBV sRz-777 15 tctgcctaaa tctct 15 16 15 DNA ArtificialSequence HBV sRz-885 16 tggagttact cgttt 15 17 15 DNA ArtificialSequence HBV m1Rz-247 17 cgcagcaggt ggagc 15 18 15 DNA ArtificialSequence HBV m1Rz-355 18 cgcgggacgc tttgt 15 19 15 DNA ArtificialSequence Pol I sRz-70 19 tcgcaatgtc atact 15 20 15 DNA ArtificialSequence Pol I sRz-353 20 tcatgctgac ccgtc 15 21 15 DNA ArtificialSequence Pol I sRz-458 21 ccatgctgca aagat 15 22 15 DNA ArtificialSequence Pol I sRz-595 22 atatcctcag ctcag 15 23 15 DNA ArtificialSequence PTEN sRz-281 23 tgaagaccaa cccac 15 24 15 DNA ArtificialSequence PTEN sRz-425 24 tttattgcag gggca 15 25 16 DNA ArtificialSequence PTEN sRz-499 25 aaaagggaga caattt 16 26 15 DNA ArtificialSequence PTEN sRz-681 26 atatattccc aattc 15 27 14 DNA ArtificialSequence PTEN sRz-774 27 gtagagttcc caca 14 28 15 DNA ArtificialSequence PTEN m1Rz-127 28 cagaaagacg aaggt 15 29 15 DNA ArtificialSequence PTEN m1Rz-151 29 ggaacaatag atgat 15 30 15 DNA ArtificialSequence PTEN m1Rz-439 30 gcaaatttta aggca 15 31 15 DNA ArtificialSequence PTEN m1Rz-760 31 gtggtgataa aagta 15 32 15 DNA ArtificialSequence PTEN m2Rz-227 32 tgagagacaa taaca 15 33 15 DNA ArtificialSequence PTEN m2Rz-304 33 tagaacttaa aaccc 15 34 15 DNA ArtificialSequence PTEN m2Rz-414 34 atttgtgcat ttatt 15 35 15 DNA ArtificialSequence PTEN m2Rz-961 35 tactcaccca caaaa 15 36 31 DNA ArtificialSequence HPV16-Dz57 36 tgtggtaagg ctagctacaa cgatttctgg g 31 37 17 DNAArtificial Sequence DNAzyme 37 cccagaaagt taccaca 17 38 17 DNAArtificial Sequence DNAzyme 38 ttaccacagt tatgcac 17 39 17 DNAArtificial Sequence DNAzyme 39 gacgtgaggt atatgac 17 40 17 DNAArtificial Sequence DNAzyme 40 catagtatat agagatg 17 41 17 DNAArtificial Sequence DNAzyme 41 attagtgagt atagaca 17 42 17 DNAArtificial Sequence DNAzyme 42 tatagtttgt atggaac 17 43 17 DNAArtificial Sequence DNAzyme 43 tataaggggt cggtgga 17 44 17 DNAArtificial Sequence DNAzyme 44 gtggaccggt cgatgta 17 45 17 DNAArtificial Sequence DNAzyme 45 gtgcttttgt gtgtctg 17 46 17 DNAArtificial Sequence DNAzyme 46 gcctctgcgt ttaggtg 17 47 17 DNAArtificial Sequence DNAzyme 47 gcctccacgt ctgcaac 17 48 17 DNAArtificial Sequence DNAzyme 48 gccgttgtgt gaaatag 17 49 17 DNAArtificial Sequence DNAzyme 49 gtggaagggt cgttgct 17 50 17 DNAArtificial Sequence DNAzyme 50 ttctcggggt cgcttgg 17 51 17 DNAArtificial Sequence DNAzyme 51 tctgcctaat catctct 17 52 17 DNAArtificial Sequence DNAzyme 52 tggagttact ctcgttt 17 53 17 DNAArtificial Sequence DNAzyme 53 tgcaatgttt caggacc 17 54 17 DNAArtificial Sequence DNAzyme 54 gccactgtgt cctgaag 17 55 17 DNAArtificial Sequence DNAzyme 55 gtggaccggt cgatgta 17 56 17 DNAArtificial Sequence DNAzyme 56 aaagatgcct ccacgtc 17 57 17 DNAArtificial Sequence DNAzyme 57 tgtgtgaaat agaaaaa 17 58 17 DNAArtificial Sequence DNAzyme 58 gtggaagggt cgttgct 17 59 17 DNAArtificial Sequence DNAzyme 59 ctttgaggat ccaacac 17 60 17 DNAArtificial Sequence DNAzyme 60 accgttgaat ccagcag 17 61 17 DNAArtificial Sequence DNAzyme 61 tgggcactat agaggcc 17 62 17 DNAArtificial Sequence DNAzyme 62 cccagaaagt taccaca 17 63 17 DNAArtificial Sequence DNAzyme 63 ttaccacagt tatgcac 17 64 17 DNAArtificial Sequence DNAzyme 64 catagtatat agagatg 17 65 17 DNAArtificial Sequence DNAzyme 65 attagtgagt atagaca 17 66 17 DNAArtificial Sequence DNAzyme 66 tatagtttgt atggaac 17 67 31 DNAArtificial Sequence DNAzyme 67 tgtggtaagg ctagctacaa cgatttctgg g 31 6831 DNA Artificial Sequence DNAzyme 68 gtgcataagg ctagctacaa cgatgtggta a31 69 31 DNA Artificial Sequence DNAzyme 69 gtcatatagg ctagctacaacgactcacgt c 31 70 31 DNA Artificial Sequence DNAzyme 70 catctctaggctagctacaa cgaatactat g 31 71 31 DNA Artificial Sequence DNAzyme 71tgtctatagg ctagctacaa cgatcactaa t 31 72 31 DNA Artificial SequenceDNAzyme 72 gttccatagg ctagctacaa cgaaaactat a 31 73 31 DNA ArtificialSequence DNAzyme 73 tccaccgagg ctagctacaa cgacccttat a 31 74 31 DNAArtificial Sequence DNAzyme 74 tacatcgagg ctagctacaa cgacggtcca c 31 7531 DNA Artificial Sequence DNAzyme 75 cagacacagg ctagctacaa cgaaaaagca c31 76 31 DNA Artificial Sequence DNAzyme 76 cacctaaagg ctagctacaacgagcagagg c 31 77 31 DNA Artificial Sequence DNAzyme 77 gttgcagaggctagctacaa cgagtggagg c 31 78 31 DNA Artificial Sequence DNAzyme 78ctatttcagg ctagctacaa cgaacaacgg c 31 79 31 DNA Artificial SequenceDNAzyme 79 agcaacgagg ctagctacaa cgaccttcca c 31 80 31 DNA ArtificialSequence DNAzyme (Dz-879) 80 gagagtaagg ctagctacaa cgatccacag t 31 81 31DNA Artificial Sequence mDz-879 81 gagagtaagc ctagctacta cgatccacag t 3182 31 DNA Artificial Sequence DNAzyme (Group L1) 82 tgccgggaggctagctacaa cgatcggggc t 31 83 31 DNA Artificial Sequence DNAzyme (GroupL2) 83 cacagaaagg ctagctacaa cgaagactga a 31 84 31 DNA ArtificialSequence DNAzyme (Group L3) 84 tatagaaggg ctagctacaa cgaagccctg c 31 8531 DNA Artificial Sequence DNAzyme (Group mL2) 85 cacagaaagc ctagctactacgaagactga a 31 86 649 DNA Mus musculus 86 caagcatagc acagagcaatgttctacttt aattactttc attttcttgt atcctcacag 60 cctagaaaat aacctgcgttacagcatcca ctcagtatcc cttgagcatg aggtgacact 120 acttaacata gggacgagatggtactttgt gtctcctgct ctgtcagcag ggcactgtac 180 ttgctgatac cagggaatgtttgttcttaa ataccatcat tccggacgtg tttgccttgg 240 ccagttttcc atgtacatgcagaaagaagt ttggactgat caatacagtc ctctgcctt 300 aaagcaatag gaaaaggccaacttgtctac ggagtcgacg gatccgggct caaatgggg 360 acaaagagat taagctcttatgtaaaattt gctgttttac ataactttaa tgaatggca 420 aagtcttgtg catgggggtgggggtggggt tagaggggaa cagctccaga tggcaaacat 480 acgcaaggga tttagtcaaacaactttttg gcaaagatgg tatgattttg taatggggta 540 ggaaccaatg aaatgcgaggtaagtatggt taatgatcta cagttattgg ttaaagaagt 600 atattagagc gagtctttctgcacacagat cacctttcct atcaacccc 649 87 13 DNA Artificial Sequenceconsensus sequence 87 aatcaannan tta 13 88 59 PRT Artificial Sequence P1pac site 88 Pro Leu Lys Ser Met Ile Ile Asp His Ser Asn Asp Gln His AlaGly 1 5 10 15 Asp His Ile Ala Ala Glu Ile Ala Glu Lys Glu Arg Val AsnAla Val 20 25 30 Val Ser Ala Ala Val Glu Asn Ala Lys Arg Gln Asn Lys ArgIle Asn 35 40 45 Asp Arg Ser Asp Asp His Asp Val Ile Thr Arg 50 55 89135 RNA Artificial Sequence ribozyme cassette 89 gcggccgcuc gagcucugaugaguccguga ggacgaaacg guacccggua ccgucagcuc 60 gaagaucuga uccgucgacggaucuagauc cguccugaug aguccgugag gacgaaacgg 120 aucugcagcg gccgc 135 9041 RNA Artificial Sequence ribozyme insert 90 gaucunnnnn nncugaugaguccgugagga caaaannnnn a 41 91 216 RNA Artificial Sequence ribozymecassette 91 aagcuuugga acccugauga guccgugagg acgaaacgau gacauucugcugaccagauu 60 cacggucagc agaaugucau cgucgguucc aggauccuug ccugaauuccaagggucugc 120 gcaacgacga cgaugaggua ccacaucguc gucguugcgc acugaugaggccgugaggcc 180 gaaacccuug acgcguuccu augcggccgc ucuaga 216 92 44 RNAArtificial Sequence ribozyme insert 92 gauccnnnnn ncugaugagu ccgugaggacgaaannnnnn nnng 44

1. A purified preparation of at least one nucleic acid molecule thatspecifically hybridizes under physiological conditions to mRNA encodingat least one viral protein associated with transformation or plasmidcopy number control or which hybridizes to a viral polyadenylationsignal or a core, pre-core or polymerase-encoding sequence, wherein atleast one sequence is selected from the group consisting of SEQ IDNOs:37-66 or a homologous sequence thereof.
 2. A purified preparation ofat least one nucleic acid molecule of claim 1, wherein said mRNA isE6/E7 mRNA.
 3. A purified preparation of at least one nucleic acidmolecule of claim 1, wherein said mRNA is papilloma viral RNA orhepatitis B viral RNA.
 4. A purified preparation of at least one nucleicacid molecule that specifically hybridizes under physiologicalconditions to a sequence selected from the group consisting of SEQ IDNOs:37-66 or a homologous sequences in a related HPV or HBV strain.
 5. Apurified preparation of at least one nucleic acid molecule of claim 1,wherein said nucleic acid molecules are DNAzymes, antisenseoligonucleotides or ribozymes.
 6. A purified preparation of at least onenucleic acid molecule of claim 1, wherein said nucleic acid moleculesare DNAzymes.
 7. A purified preparation of at least one nucleic acidmolecule of claim 1, wherein said nucleic acid molecules are antisenseoligonucleotides.
 8. A purified preparation of at least one nucleic acidmolecule of claim 1, wherein said nucleic acid molecules are ribozymes.9. A purified preparation of at least one nucleic acid molecule of claim1, wherein said nucleic acid molecules are resistant to nucleasedegradation.
 10. A purified preparation of at least one nucleic acidmolecule of claim 9, wherein said nucleic acid molecules are modified attheir 3′ ends, are resistant to nuclease degradation and have a 3′-3′inverted T at their 3′ ends.
 11. A pharmaceutical composition comprisingany of the nucleic acid molecules of claim 1 and a pharmaceuticallyacceptable carrier.
 12. A method of treating papilloma virus-inducedconditions or hepatitis virus-induced conditions comprisingadministering to a subject a pharmaceutical compositions of claim 11.13. A method of claim 12, wherein said administration comprises topicalapplication to the cervix.
 14. A method of claim 12, wherein saidadministration comprises topical application to the epidermis.
 15. Apharmaceutical composition of claim 11, wherein said nucleic acidmolecules are formulated as a cosmetic formulation.
 16. A pharmaceuticalcomposition of claim 11, wherein said nucleic acid molecules areformulated in an amount sufficient to produce a cytotoxic or cytostaticeffect in cells infected with papilloma virus or hepatitis B virus. 17.A pharmaceutical composition of claim 16, wherein said cells aretransformed by a papilloma virus.
 18. A pharmaceutical composition ofclaim 16, wherein said cells are transformed by a hepatitis B virus. 19.A pharmaceutical composition of claim 16, wherein said nucleic acidmolecules are formulated for topical administration.
 20. Apharmaceutical composition of claim 19, wherein said nucleic acids areformulated into an ointment, salve, gel, cream or lotion.
 21. Apharmaceutical composition of claim 11, wherein said nucleic acidmolecules are formulated into a liposome preparation or a lipidpreparation.
 22. A method of claim 12, wherein said papillomavirus-induced condition is selected from the group consisting of warts,cervical dysplasia, cervical carcinoma, carcinoma in situ and laryngealpapilloma.
 23. A method of claim 22, wherein said administration is toepithelial cells.
 24. A method of claim 23, wherein said epithelialcells are selected from the group consisting of squamous epithelia,cutaneous epithelia and mucosal epithelia.
 25. The pharmaceuticalcomposition of claim 11 wherein said one or more nucleic acids areformulated for delivery in a liposome.
 26. The pharmaceuticalcomposition of claim 25 wherein said liposome is capable oftissue-specific uptake in the liver.
 27. The pharmaceutical compositionof claim 26 wherein said liposome is modified using asialofetuin or oneor more sugars.